title: Genomewide_ERHoxa9_Day6_all_all_rep2

data_processing:
For data analysis, FASTQ files were demultiplexed and barcode sequences were matched to the master list of CRISPRi barcodes; only perfect matches were retained. Raw counts were subsequently processed using MAGECK to obtain sgRNA-level and gene-level scores and their corresponding p-values (Table S3 of accompanying manuscript).
Assembly: hg38, mm10
Supplementary files format and content: Raw counts for each sgRNA for each cell fraction.

source_name: ER-Hoxa9 dCas9-KRAB cells

growth_protocol: THP-1 and K562 cells were grown in RPMI (Corning, 15-040-CV) with 10% dialyzed fetal bovine serum (Sigma, F0392), 4mM glutamine, and 1% penicillin/streptomycin (Corning, 45000-652). ER-Hoxa9 cells were grown in RPMI (Corning, 15-040-CV) with 10% dialyzed fetal bovine serum (Sigma, F0392), 4mM glutamine, and 1% penicillin/streptomycin (Corning, 45000-652), supplemented with 0.5uM beta-estradiol (Sigma, E2758; made from a 10mM stock dissolved in 100% ethanol) and stem cell factor (SCF) from conditioned media generated from a Chinese hamster ovary (CHO) cell line that stably secretes SCF (Sykes et al. Cell 2016; this conditioned media was added at a concentration of 1%).

extract_protocol:
CRISPRi screens were performed using ER-Hoxa9, THP-1, or K562 dCas9-KRAB cells generated as above. For the genome-wide screens, libraries with 5 sgRNAs per gene (mCRISPRi-v2; the “top5” library from Addgene #1000000092) or 10 sgRNAs per gene (hCRISPRi-v2; both the “top5” and “supp5” libraries from Addgene #1000000090) were prepared as described in Palmer et al.119 with several modifications. Briefly, each plasmid library was electroporated into MegaX cells, maintaining at least 2000X representation, and plasmid was maxiprepped; the libraries were sequenced as described below to confirm even representation of guides. LentiX cells were transfected with plasmid as described above, and 48 hours later supernatant was collected and frozen in aliquots at -80ºC. ER-Hoxa9 and THP-1 cells were always maintained between 250,000 and 1 million cells/ml, and two biological replicates were performed starting at the lentiviral infection step. 200 million cells (THP-1) or 150 million cells (ER-Hoxa9) were infected for 24 hours with lentiviral supernatant with 8 μg/ml polybrene, achieving an MOI of between 0.2 and 0.35. Lentiviral media was then replaced with fresh media, and 24 hours later, 2.5 μg/ml puromycin (ER-Hoxa9) or 1 μg/ml puromycin (THP-1) was added to select sgRNA-expressing cells. Media was exchanged and fresh puromycin was added 24 and 48 hours afterwards. Four days after puromycin treatment, viability was >90% and BFP positivity was >90% for both replicates. At this point, puromycin was removed and 200 million (THP-1) or 50 million cells (ER-Hoxa9) were treated with DMSO or BRQ (500 nM for THP-1 cells). 48 hours later, cells were concentrated to 20 million cells/ml in fresh media and stained with antibodies and propidium iodide, and at least 15 million (THP-1) or 10 million (ER-Hoxa9) BFP-positive live cells from both the top and bottom 25% of flow marker expression (CD11b or CD14 for THP-1; CD11b or GFP for ER-Hoxa9) were sorted using a BD FACSAria, pelleted, and frozen at -80ºC for later processing. Aliquots of 15-20 million unsorted cells before and after treatment were also pelleted and frozen as controls for guide enrichment or dropout.  For the sublibrary screen, the top and bottom 300 hits of the THP-1 BRQ CD11b screen were selected, and all ten sgRNAs from the hCRISPRi-v2 library were incorporated into the sublibrary, along with ten sgRNAs for 894 randomly chosen genes and 87 hand-curated genes as well as 490 control sgRNAs, and oligos were synthesized as a pool (Twist Biosciences) (Table S4). Oligos were cloned into pU6-sgRNA EF1Alpha-puro-T2A-BFP (Addgene plasmid #60955) using methods described in Horlbeck et al. 2014120; briefly, oligos were amplified and digested with BstXI and BlpI, the 33bp insert containing the sgRNA sequence was size selected on a 10% PAGE gel and purified, and ligation into the linearized backbone was performed at a 1:1 molar ratio of insert to vector. Ligated plasmids were electroporated into MegaX cells at approximately 5000X library coverage, maxiprepped, and transfected into LentiX cells. 25 million THP-1 and K562 dCas9-KRAB cells were infected for 24 hours in duplicate with lentivirus and 8 µg/ml polybrene (MOI of between 0.25 and 0.35 for both cell lines), lentiviral media was replaced with fresh media, and 24 hours later puromycin selection was started at a concentration of 1 µg/ml. Cells were maintained at a concentration of between 250,000 and 1 million cells/ml and with a minimum of 500X library coverage throughout the selection, and puromycin was refreshed as above. After four days of selection, >90% of cells were BFP positive. 8 million K562 or THP-1 cells were treated with DMSO and 16 million THP-1 cells were treated with 500 nM BRQ for 48 hours (all screens performed in duplicate). 48 hours later, cells were concentrated to 20 million/ml and stained with antibodies and propidium iodide, and the top and bottom 25% of CD235a-expressing K562 cells or the top and bottom 25% of CD11b or CD14-expressing THP-1 cells were sorted and frozen at -80ºC for later processing. At least 2 million BFP+ live cells were collected per gate to achieve a minimum of 100X library coverage. Aliquots of 5 million unsorted cells before and after each treatment were also pelleted and frozen as controls for guide enrichment or dropout.
To obtain amplicons for sequencing, genomic DNA from each cell pellet was extracted using the Machery Nagel Nucleospin Blood L kit (for genome-wide screens) or the Qiagen DNA Blood Mini kit (for sublibrary screens) following the manufacturer’s instructions except for an overnight incubation at 70°C and elution of DNA into 200 μl 10mM Tris (pH 8.5). Guides were then PCR amplified using custom i7 and custom i5 primers indexed for each sample, with reactions split such that each 50 μl PCR reaction contained no more than 2.5 μg DNA in a 20 μl volume. For the THP-1 genome wide CRISPRi screens, each library was amplified using one of the p5 primers oBD370-375 and one of the p7 primers oBD367-368 (Table S5). For the ER-Hoxa9 genome-wide CRISPRi screens and all sublibrary screens, each library was amplified using one of the p5 primers oSV104-111 and one of the p7 primers oSV112-119 (Table S5). PCR was performed using the NEBNext Ultra Q5 2X Master Mix with the following cycling parameters: initial denaturation at 98°C for 2 minutes, then 24 cycles of denaturation at 98°C for 15 seconds and annealing/extension at 65°C for 75 seconds, then a final extension of 65°C for 5 minutes. All reactions for each pellet were pooled, and successful amplification was verified by running 10 μl of the PCR on an agarose gel to obtain an expected band between 250 and 260bp. 200 μl of the PCR reaction (roughly corresponding to 1 μg of amplicon) was then purified using a double-sided selection with AMpure XP beads. First, to remove genomic DNA, 0.5X volume of beads (100 μl) was added to the PCR reaction, mixed well, and incubated at room temperature for 10 minutes, and beads were placed on a magnet and supernatant was saved. Second, to remove primer dimers, 135 μl beads were added to 290 μl of supernatant (final bead volume of 1.2X), mixed well, and incubated at room temperature for 10 minutes, and beads were placed on a magnet and supernatant was discarded. DNA was then eluted from the beads using 20 μl of 0.1X TE (pH 8.5), quantified using Qubit and Bioanalyzer, and pooled for 1x75 sequencing on an Illumina NextSeq 500. For the THP-1 genome-wide CRISPRi screen, we used an equimolar mix of custom read 1 primers, numbered oBD379-384 (Table S5). For the ER-Hoxa9 genome wide screens and all sublibrary screens, we used primer oBD582 (Table S5).

value: ER-Hoxa9 dCas9-KRAB cells


title: Genomewide_ERHoxa9_Day6_all_all_rep1

status: Public on May 04 2024

type: SRA

city: Cambridge

first: Brian

last: Do

state: MA

address: 500 Main St

department: Biology

country: USA

institute: MIT

library_source: genomic

molecule: genomic DNA

organism: Mus musculus

tag: cell line

tag: treatment

value: DMSO


title: iPS201-RPE, 6 weeks, rep1, cell132

city: Suita

first: Katsuyuki

last: Shiroguchi

state: Osaka

address: 6-2-3 Furuedai

department: Center for Biosystems Dynamics Research (BDR)

country: Japan

institute: RIKEN

library_source: transcriptomic

data_processing:
Basecalls performed by Illumina Hiseq
Sequenced reads were trimmed for adaptor sequence, and were sorted by indexes using bcl2fastq
Reads were aligned to the gh38 genome assebly using STAR
Mapped reads seperated by genes, and the reads mapped for each gene were clusterd by molecular barcode
The number of cluteres for each gene was counted, which provides the number of molecules detected for each gene
Assembly: gh38
Supplementary files format and content: The calculated copy number of RNA moelcules per cell for each gene is listed.

molecule: total RNA

organism: Homo sapiens

source_name: iPSC derived RPE cell

extract_protocol:
RNAs from cell lysate in each well was directly used for library preparation without purification
Libraries were prepared by using molecular barcoding technique. First, for control, the same amount of spike-in RNAs, a known number of External RNA Controls Consortium (ERCC) RNA molecules, was added into every tube which contains an isolated cell. Then, the cells were lysed and RNA molecules were fragmentated by temperature elevation. cDNAs were generated by reverse transcription using a reverse transcription primer, in which the first common primer sequence, molecular barcode, fourteen T bases, and one V (A or G or C) base are tandemly arranged from 5' end. In the reverse transcription, the second common primer sequence was also attached to 3' end of each generated cDNA molecule by template switching. Subsequently, cDNAs were amplified by PCR using two primers; one primer contains an illumina adapter sequence, sample index, and the first common primer sequence, and the other primer has another illumina adapter sequence, sample index, and the second common primer sequence.

treatment_protocol: Single cells were isolated in wells respectively using Automated Live imaging and cell Picking System (ALPS)

tag: tissue

value: iPSC derived RPE cell

tag: cell type

value: RPE

tag: rpe culture_period

value: 6 weeks


title: Genomewide_ERHoxa9_Day8_DMSO_GFP_low_rep2


title: ERHoxa9_sgNTC_r2

data_processing:
Mouse and human data were trimmed using Cutadapt (v3.4) (with parameters -m 20 -q 20 -a CTGTCTCTTATA -A CTGTCTCTTATA), then aligned to the mm10 and hg38 genomes with Bowtie2 v2.4.2 (with parameters --dovetail -I 10 -X 1000 --very-sensitive).
Reads in the mitochondrial genome and unannotated chromosomes as well as the UCSC blacklist were removed, then duplicates were removed with samtools markdup (v1.1.2), and the insert size distribution was calculated using Picard CollectInsertSizeMetrics (v2.25.2) and validated to contain nucleosomal peaks. BED file was then constructed with each mate pair as a separate read using bedtools bamtobed (v2.3.0), and peaks were called using MACS2 callpeak (v2.2.7.1) (with parameters --nomodel --shift -100 --extsize 200 --keep-dup all --call-summits). Note that the MACS2 command shifts each read by 100 base pairs towards the 5’ end and extends the read length to 200 base pairs, thus treating the 5’ end of the original read (the Tn5 cut site) as the center of a new “fragment” that is then used for peak calling and other downstream analysis.
To normalize tracks, reads overlapping shared peaks were counted for each BAM file using featureCounts (with parameters -M -O) and normalization factors were calculated from these counts using the calcNormFactors function in the edgeR package (v3.32.1) in R. Visualization was performed using the bedGraphToBigWig utility and the igv-jupyter package in Python.
Assembly: mm10
Supplementary files format and content: bigwig

source_name: ER-Hoxa9 dCas9-KRAB

growth_protocol: Cells were grown in RPMI (Corning, 15-040-CV) with 10% dialyzed fetal bovine serum (Sigma, F0392), 4mM glutamine, and 1% penicillin/streptomycin (Corning, 45000-652), supplemented with 0.5uM beta-estradiol (Sigma, E2758; made from a 10mM stock dissolved in 100% ethanol) and stem cell factor (SCF) from conditioned media generated from a Chinese hamster ovary (CHO) cell line that stably secretes SCF (Sykes et al. Cell 2016; this conditioned media was added at a concentration of 1%).

extract_protocol:
To generate ATAC-seq libraries for 63.3 cells, 50,000 cells were used and libraries were constructed as previously described (Buenrostro et al., 2013; Cheloufi et al., 2015). Briefly, cells were washed in PBS twice, counted and nuclei were isolated from 100,000 cells using 100 μl hypotonic buffer (10 mM Tris pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% NP40) to generate two independent transposition reactions. Nuclei were split in half and treated with 2.5 μl Tn5 Transposase (Illumina) for 30 mins. at 37°C.
DNA from transposed nuclei was then isolated and PCR-amplified using barcoded Nextera primers (illumina). Library QC was carried out using high sensitivity DNA bioanalyzer assay and Qubit measurement and sequenced using paired-end sequencing (PE50) on the Illumina HiSeq 2500 platform.

value: ER-Hoxa9 dCas9-KRAB

tag: genotype

value: sgNTC


title: iPS201-RPE, 6 weeks, rep1, cell133


title: iPS201-RPE, 6 weeks, rep1, cell127


title: iPS201-RPE, 6 weeks, rep1, cell130


title: ERHoxa9_sgNTC_r1


title: iPS201-RPE, 6 weeks, rep1, cell126


title: iPS201-RPE, 6 weeks, rep1, cell125


title: Genomewide_ERHoxa9_Day8_DMSO_CD11b_high_rep2


title: iPS201-RPE, 6 weeks, rep1, cell129


title: Genomewide_ERHoxa9_Day8_DMSO_GFP_high_rep1


title: iPS201-RPE, 6 weeks, rep1, cell128


title: Genomewide_ERHoxa9_Day8_DMSO_GFP_high_rep2


title: Genomewide_ERHoxa9_Day8_DMSO_GFP_low_rep1


title: Genomewide_ERHoxa9_Day8_DMSO_CD11b_low_rep1


title: iPS201-RPE, 6 weeks, rep1, cell122


title: Genomewide_ERHoxa9_Day8_DMSO_CD11b_low_rep2


title: iPS201-RPE, 6 weeks, rep1, cell123


title: iPS201-RPE, 6 weeks, rep1, cell121


title: iPS201-RPE, 6 weeks, rep1, cell137


title: iPS201-RPE, 6 weeks, rep1, cell120


title: Genomewide_ERHoxa9_Day8_DMSO_CD11b_high_rep1


title: iPS201-RPE, 6 weeks, rep1, cell119


title: MG_IFN_Blood_IFN4_Ly6CpSca1n_7

city: Rehovot

first: Steffen

last: Jung

address: 234 Herzl Street

department: Department of Immunology

country: Israel

institute: Weizmann Institute of Science

data_processing:
Data analysis was performed using the UTAP transcriptome analysis pipeline 78.Raw reads were trimmed using cutadapt with the parameters: -a AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC -a ‘‘A–times 2 -u 3 -u 3 -q 20 -m 25). Reads were mapped to the genome (mm10, Gencode annotation version 10.0) using STAR (v2.4.2a) with the parameters –alignEndsType EndToEnd, –outFilterMismatchNoverLmax 0.05, –twopassMode Basic, –alignSoftClipAtReferenceEnds No. The pipeline quantifies the 30 of Gencode annotated genes (The 30 region contains 1,000 bases upstream of the 30 end and 100 bases downstream). UMI counting was done after marking duplicates (in-house script) using HTSeq-count in union mode. Only reads with unique mapping were considered for further analysis, and genes having minimum 5 reads in at least one sample were considered. Gene expression levels were calculated and normalized using DESeq2 with the parameters: betaPrior=True, cooksCutoff=FALSE, independentFilter- ing=FALSE. Batch correction was done using the sva (3.26.0) R when batch adjustments were required. Raw p-values were adjusted for multiple testing, using the procedure of Benjamini and Hochberg. Differentially expressed genes were selected with absolute fold change (log2)>1, and adjusted p-value <0.05. Visualization of gene expression heatmaps was done using Gene-e, using log normalized values. Clustering was applied on log2 transformed and standardized expression values, using the k-means algorithm (Euclidian method). For Gene Ontology (GO) term analysis, Metascape was used 57, and all heatmaps were made by Morpheus, https://software.broadinstitute.org/morpheus.
Assembly: mm10, Gencode annotation version 10.0
Supplementary files format and content: txt files, raw counts

molecule: polyA RNA

source_name: blood

extract_protocol: Peripheral blood or lung cells were isolated as previously described and sorted into 30 ul of lysis/binding buffer (home-made or Life Technologies) and stored at -80°C. mRNA was then captured with Dynabeads oligo(dT) (Life Technologies) according to the manufacturer’s guidelines. A bulk variation of MARSseq 77 was used for preparation of RNAseq libraries. Briefly, RNA was first reverse-transcribed with MARSseq barcoded RT primers with the Affinity Script kit (Agilent). Reverse transcription was analyzed by qRT-PCR and samples with a similar CT were pooled. Each pool was treated with Exonuclease I (NEB) for 30’ at 37°C. Double-stranded DNA was generated with the NEB second strand synthesis kit at 16°C for 2h.), Subsequently, in vitro transcription was performed over-night with the T7 High Yield RNA polymerase IVT kit (NEB) for 37°C overnight. Following IVT, the DNA template was removed with Turbo DNase I (Ambion) at 37°C for 15’. Amplified RNA was fragmented by incubation at 70°C for 3’ in Zn2+ RNA fragmentation reagent (Ambion). RNA was then ligated to the MARSseq ligation adaptor with T4 RNA Ligase I (NEB) at 22°C for 2h. Ligated product was reverse-transcribed using the Affinity Script RT enzyme (Agilent) and a primer complementary to the ligated product. The library was completed and amplified through a nested PCR reaction with P5_Rd1 and P7_Rd2 primers and PCR ready mix (Kappa Biosystems). Library concentrations was measured with a Qubit fluorometer (Life Technologies) and mean molecule size was determined with a 2200 TapeStation instrument. RNAseq libraries were sequenced using the Illumina NextSeq 500.

value: blood

tag: strain background

value: classical monocyte

value: WT

value: recombinant IFNg


title: mmCRISPRi_library1_pcr

source_name: Addgene 83987

value: Addgene 83987


title: MG_IFN_Blood_IFN3_Ly6CpSca1n_7


title: MG_IFN_Blood_IFN4_Ly6CpSca1p_8


title: MG_IFN_Blood_IFN3_Ly6CpSca1p_8


title: iPS201-RPE, 6 weeks, rep1, cell118


title: iPS201-RPE, 6 weeks, rep1, cell117


title: MG_IFN_Blood_IFN2_Ly6CpSca1n_7


title: MG_IFN_Blood_IFN1_Ly6CpSca1p_8


title: iPS201-RPE, 6 weeks, rep1, cell116


title: iPS201-RPE, 6 weeks, rep1, cell124


title: MG_IFN_Blood_IFN2_Ly6CpSca1p_8


title: iPS201-RPE, 6 weeks, rep1, cell111


title: MG_IFN_Blood_Con3_Ly6CpSca1n_3

value: PBS


title: MG_IFN_Blood_Con4_Ly6CpSca1n_3


title: iPS201-RPE, 6 weeks, rep1, cell114


title: iPS201-RPE, 6 weeks, rep1, cell115


title: iPS201-RPE, 6 weeks, rep1, cell110


title: iPS201-RPE, 6 weeks, rep1, cell113


title: MG_IFN_Blood_IFN1_Ly6CpSca1n_7


title: iPS201-RPE, 6 weeks, rep1, cell109


title: iPS201-RPE, 6 weeks, rep1, cell107


title: iPS201-RPE, 6 weeks, rep1, cell108


title: MG_IFN_Blood_Con2_Ly6CpSca1n_3


title: ST_NeuDCMo_NeuMo_2

value: GMP-derived classical monocyte


title: ST_NeuDCMo_NeuMo_3


title: iPS201-RPE, 6 weeks, rep1, cell106


title: ST_NeuDCMo_NeuMo_1


title: iPS201-RPE, 6 weeks, rep1, cell103


title: ST_NeuDCMo_DN_3


title: ST_NeuDCMo_DN_4


title: iPS201-RPE, 6 weeks, rep1, cell102


title: ST_NeuDCMo_DN_2


title: iPS201-RPE, 6 weeks, rep1, cell101


title: ST_NeuDCMo_DN_5


title: ST_NeuDCMo_DN_1


title: iPS201-RPE, 6 weeks, rep1, cell112


title: iPS201-RPE, 6 weeks, rep1, cell100


title: iPS201-RPE, 6 weeks, rep1, cell98


title: ST_NeuDCMo_DCMo_5

value: MDP-derived classical monocytes


title: ST_NeuDCMo_NeuMo_4


title: iPS201-RPE, 6 weeks, rep1, cell97


title: iPS201-RPE, 6 weeks, rep1, cell99


title: iPS201-RPE, 6 weeks, rep1, cell96


title: ST_NeuDCMo_DCMo_2


title: iPS201-RPE, 6 weeks, rep1, cell105


title: ST_NeuDCMo_DCMo_1


title: ST_DR_GFP_4

source_name: bone marrow

value: bone marrow


title: iPS201-RPE, 6 weeks, rep1, cell95


title: iPS201-RPE, 6 weeks, rep1, cell104


title: iPS201-RPE, 6 weeks, rep1, cell94


title: ST_NeuDCMo_DCMo_3


title: ST_NeuDCMo_DCMo_4


title: ST_DR_GFP_2


title: ST_DR_GFP_1


title: iPS201-RPE, 6 weeks, rep1, cell93


title: iPS201-RPE, 6 weeks, rep1, cell92


title: ST_DR_GFP_3


title: iPS201-RPE, 6 weeks, rep1, cell91


title: ST_DR_DP_4


title: iPS201-RPE, 6 weeks, rep1, cell90


title: iPS201-RPE, 6 weeks, rep1, cell89


title: ST_DR_DP_2


title: iPS201-RPE, 6 weeks, rep1, cell88


title: iPS201-RPE, 6 weeks, rep1, cell87


title: iPS201-RPE, 6 weeks, rep1, cell86


title: ST_DR_DP_1


title: ST_Adopt_Transf_Neu3

source_name: lung

value: lung

value: GMP-derived lung macrophage


title: iPS201-RPE, 6 weeks, rep1, cell85


title: ST_Adopt_Transf_Neu2


title: ST_Adopt_Transf_DCT2

value: MDP-derived lung macrophage


title: iPS201-RPE, 6 weeks, rep1, cell84

value: iPSC derived RPE cell


title: ST_Adopt_Transf_Neu1


title: ST_Adopt_Transf_DCG2


title: iPS201-RPE, 6 weeks, rep1, cell83

value: RPE


title: ST_Adopt_Transf_DCG1


title: ER-Hoxa9_E2wd24_H3K27ac

description: CUT&RUN

data_processing:
Mouse and human data were mapped to mm10 and hg38 genomes, respectively, using Bowtie2 v2.4.2 (with parameters --dovetail -I 10 -X 700 --very-sensitive-local --local)
BAM files were then normalized to 10 million total reads.
Supplementary files format and content: Bigwig files for each sample

source_name: ER-Hoxa9 mouse GMP cells

extract_protocol:
Cells were stained with a final concentration of 1 ng/ml DAPI in growth media, and 1 million live cells were sorted using a BD FACSAria and placed on ice. CUT&RUN was then performed using the Epicypher CUTANA CUT&RUN kit, using the indicated number of cells, antibody, and concentration of antibody.
Library construction was performed using the NEBNext Ultra II Library Preparation Kit following the indicated protocol, and each library was indexed using NEBNext® Multiplex Oligos for Illumina. For PU.1, the library construction protocol was modified to generate shorter fragments per https://www.protocols.io/view/library-prep-for-cut-amp-run-with-nebnext-ultra-ii-wvgfe3w. Libraries were quantified using Qubit and Bioanalyzer and pooled for sequencing using an Illumina NextSeq 500.
OTHER: CUT&RUN

value: Estrogen withdrawal

tag: antibody

value: H3K27ac, Millipore MABE647

tag: treatment duration

tag: cell number

tag: antibody concentration

tag: genome build


title: ER-Hoxa9_BRQ24_H3K27ac

description: CUT&RUN

value: Brequinar 1uM


title: iPS201-RPE, 6 weeks, rep1, cell79

value: 6 weeks


title: iPS201-RPE, 6 weeks, rep1, cell81


title: ER-Hoxa9_DMSO24_H3K27ac

description: CUT&RUN

value: DMSO


title: iPS201-RPE, 6 weeks, rep1, cell80


title: iPS201-RPE, 6 weeks, rep1, cell77


title: iPS201-RPE, 6 weeks, rep1, cell82


title: ER-Hoxa9_DMSO_IgG

description: CUT&RUN

value: IgG, Epicypher 13-0042


title: ER_cst8644_E2wd24

description: CUT&RUN

value: ER, CST 8644


title: ST_DR_DP_3


title: iPS201-RPE, 6 weeks, rep1, cell74


title: ER_cst8644_E2wd8

description: CUT&RUN


title: iPS201-RPE, 6 weeks, rep1, cell78


title: iPS201-RPE, 6 weeks, rep1, cell72


title: iPS201-RPE, 6 weeks, rep1, cell75


title: ER-Hoxa9_dCas9KRAB_sgSPI1-2_BRQ_rep4

data_processing:
Mouse and human data were mapped to mm10 and hg38 genomes, respectively, using STAR v2.7.8a and default parameters.
Reads that mapped to transcripts were counted using featureCounts v2.0.1 (with parameters -M -O --fraction --primary).
Supplementary files format and content: A tab-delimited file containing raw counts generated from featureCounts.

source_name: ER-Hoxa9 mouse GMP cells (dCas9-Zim3-KRAB; sgSpi1-2)

growth_protocol: Cells were grown in RPMI (Corning, 15-040-CV) with 10% dialyzed fetal bovine serum (Sigma, F0392), 4mM glutamine, and 1% penicillin/streptomycin (Corning, 45000-652).

extract_protocol:
1 million live cells were sorted using a BD FACSAria and placed on ice. Immediately following the sort, cells were centrifuged, pelleted, and flash frozen in liquid nitrogen and placed at -80C. Pellets were processed in groups of 24 using the Zymo RNA Miniprep Kit and quantified using the Invitrogen Quant-it RNA kit. RNA was then diluted to a concentration of 2 ng/ul and 5ul of each sample was transferred to a 96 well plate.
Poly-A enrichment and cDNA synthesis were performed in parallel as described in Soumilion et al. 2014, followed by library preparation using the Nextera XT kit and paired-end sequencing on an Illumina NextSeq 500.


title: ER_cst8644_BRQ8

description: CUT&RUN


title: iPS201-RPE, 6 weeks, rep1, cell71


title: iPS201-RPE, 6 weeks, rep1, cell73


title: ER_cst8644_DMSO

description: CUT&RUN


title: iPS201-RPE, 6 weeks, rep1, cell70


title: iPS201-RPE, 6 weeks, rep1, cell69


title: ER-Hoxa9_dCas9KRAB_sgSPI1-2_BRQ_rep1


title: ER-Hoxa9_dCas9KRAB_sgSPI1-2_BRQ_rep3


title: ER-Hoxa9_dCas9KRAB_sgSPI1-2_BRQ_rep2


title: iPS201-RPE, 6 weeks, rep1, cell67


title: ER-Hoxa9_dCas9KRAB_sgSPI1-2_DMSO_rep4


title: ER-Hoxa9_dCas9KRAB_sgSPI1-2_DMSO_rep3


title: iPS201-RPE, 6 weeks, rep1, cell66


title: ER-Hoxa9_dCas9KRAB_sgSPI1-2_DMSO_rep1


title: ER-Hoxa9_dCas9KRAB_sgSPI1-2_DMSO_rep2


title: iPS201-RPE, 6 weeks, rep1, cell76


title: iPS201-RPE, 6 weeks, rep1, cell63


title: iPS201-RPE, 6 weeks, rep1, cell65


title: iPS201-RPE, 6 weeks, rep1, cell64


title: iPS201-RPE, 6 weeks, rep1, cell62


title: ER-Hoxa9_dCas9KRAB_sgSPI1-1_BRQ_rep3

source_name: ER-Hoxa9 mouse GMP cells (dCas9-Zim3-KRAB; sgSpi1-1)


title: ER-Hoxa9_dCas9KRAB_sgSPI1-1_BRQ_rep1


title: ER-Hoxa9_dCas9KRAB_sgSPI1-1_BRQ_rep2


title: ER-Hoxa9_dCas9KRAB_sgSPI1-1_BRQ_rep4


title: ER_cst8644_BRQ24

description: CUT&RUN


title: ER-Hoxa9_dCas9KRAB_sgSPI1-1_DMSO_rep4


title: iPS201-RPE, 6 weeks, rep1, cell59


title: iPS201-RPE, 6 weeks, rep1, cell68


title: iPS201-RPE, 6 weeks, rep1, cell58


title: ER-Hoxa9_dCas9KRAB_sgSPI1-1_DMSO_rep2


title: ER-Hoxa9_dCas9KRAB_sgSPI1-1_DMSO_rep1


title: iPS201-RPE, 6 weeks, rep1, cell55


title: iPS201-RPE, 6 weeks, rep1, cell57


title: ER-Hoxa9_dCas9KRAB_sgNTC2_BRQ_rep3

source_name: ER-Hoxa9 mouse GMP cells (dCas9-Zim3-KRAB; sgNTC)


title: ER-Hoxa9_dCas9KRAB_sgNTC2_BRQ_rep4


title: iPS201-RPE, 6 weeks, rep1, cell56


title: ER-Hoxa9_dCas9KRAB_sgNTC2_BRQ_rep2


title: iPS201-RPE, 6 weeks, rep1, cell54


title: iPS201-RPE, 6 weeks, rep1, cell60


title: iPS201-RPE, 6 weeks, rep1, cell52


title: ER-Hoxa9_dCas9KRAB_sgNTC2_BRQ_rep1


title: iPS201-RPE, 6 weeks, rep1, cell51


title: iPS201-RPE, 6 weeks, rep1, cell50


title: iPS201-RPE, 6 weeks, rep1, cell61


title: ER-Hoxa9_dCas9KRAB_sgNTC2_DMSO_rep2


title: ER-Hoxa9_dCas9KRAB_sgNTC2_DMSO_rep3


title: iPS201-RPE, 6 weeks, rep1, cell49


title: ER-Hoxa9_dCas9KRAB_sgSPI1-1_DMSO_rep3


title: iPS201-RPE, 6 weeks, rep1, cell53


title: ER-Hoxa9_dCas9KRAB_sgNTC2_DMSO_rep1


title: iPS201-RPE, 6 weeks, rep1, cell48


title: iPS201-RPE, 6 weeks, rep1, cell43


title: 633_MULTIseq_run1_with_barcodes

description: scRNAseq

data_processing:
FASTQ files from both runs were first concatenated. Note that run 1 and run 2 come from the same sample, but run 1 does not have MULTI-seq barcodes spiked in, so demultiplexing is performed on the run 2 FASTQ files.
Concatenated FASTQ files were then mapped to the mouse cDNA and intronic transcriptomes with the 10x v3 whitelist  (all files available at https://github.com/pachterlab/MBGBLHGP_2019/releases) using Kallisto v0.46 and BUStools v0.39.4.
Samples were then demultiplexed using the deMULTIplex package (v1.0.2) in R (https://github.com/chris-mcginnis-ucsf/MULTI-seq). Annotations for each cell barcode are in demultiplex_annotation.csv.
Barcodes correspond to samples as tabulated in 633_MULTIseq_barcodes_to_sample.csv.
Supplementary_files_format_and_content: Tab-delimited files with barcode annotations and Cell Ranger-compatible MTX files, genes, and barcodes. The file s.mtx.gz contains counts for exons of genes. The file u.mtx.gz contains counts for introns of genes. The files s.barcodes.txt.gz and s.genes.txt.gz can be used for both matrices. The file demultiplex_annotation.csv contains barcode annotations for each gene.

source_name: 63.3 mouse AML cells

extract_protocol:
Cells were lysed as indicated in the 10X Genomics protocol.
Library construction proceeded as indicated in the 10X Genomics scRNAseq v3 protocol except as follows (adapted from McGinnis et al. 2019). During cDNA amplification, 1ul of a 2.5uM MULTI-seq primer (5'-CTTGGCACCCGAGAATTCC-3') was added to the master mix. After the 0.6X SPRI clean-up, the supernatant was saved for barcode extraction rather than discarded. To the 60ul supernatant, 260ul SPRI beads and 180ul 100% isopropanol were added for a final ratio of 1.8X beads, pipetted up and down 10x, and incubated at room temperature for 5 minutes. The tube was then placed on a magnetic rack for 5 minutes, supernatant discarded, beads washed 2x with 500ul 80% ethanol, and eluted with 50ul buffer EB. This SPRI purification step was repeated once more to further purify the barcode fraction. The barcode fraction was quantified using Qubit, and then library preparation PCR was performed using the Kapa HiFi HotStart ReadyMix with 3.5ng barcode DNA, 2.5ul 10uM universal I5 primer, and 2.5ul 10uM RPI1 primer (5'-CAAGCAGAAGACGGCATACGAGATATCACGGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA-3') in a 50ul reaction for 8-12 cycles. The PCR product was purified using a final SPRI bead concentration of 1.6X, eluted, and quantified on a Bioanalyzer; a peak between 175 and 200bp was expected. The barcode library was mixed 1:10 with the transcript library and sequenced on a NextSeq 500.

treatment_protocol: For brequinar treatments, 1 million cells were treated with Brequinar 2uM for 6, 12, 24, or 48h. For estrogen withdrawal, 1 million cells were washed twice with PBS and then resuspended in media without estrogen for 6, 12, 24, or 48h. To barcode each sample, MULTI-seq (McGinnis et al. 2019) was used; the barcode for each sample is included in the supplementary files. Briefly, for each condition, 400,000 cells were rinsed twice in PBS and resuspended in 180ul PBS. 20ul of a 10X stock of anchor:barcode (per sample: 0.88ul anchor, 0.44ul 100uM barcode, and 20.7ul PBS) was added to each sample, pipetted gently to mix, and incubated on ice for 5 minutes. Next, 20ul of a 10X stock of coanchor (per sample: 0.88ul co-anchor and 21.2ul PBS) was added to each sample, pipetted gently to mix, and incubated on ice for 5 minutes. 700ul ice cold 1% BSA in PBS was then added to each sample, and cells were centrifuged at 4C for 5 minutes. The supernatant was aspirated and the wash was repeated 2X with ice cold BSA in PBS. After the last wash, cells were resuspended in 300ul PBS/BSA, filtered through a cell strainer, and counted using a Nexcelom cell counter. Cells were adjusted to a concentration of 1 million/ml using ice cold BSA in PBS, and 50ul of each sample was pooled into one tube and placed on ice. Cells were then loaded into the 10X Genomics microfluidics chip for processing and scRNAseq.

value: 63.3 mouse AML cells


title: 63.3_BRQ_H3K4me1

description: CUT&RUN

data_processing:
Library strategy: CUT&RUN
Mouse and human data were mapped to mm10 and hg38 genomes, respectively, using Bowtie2 v2.4.2 (with parameters --dovetail -I 10 -X 700 --very-sensitive-local --local)
BAM files were then normalized to 10 million total reads.
Genome_build: mm10
Genome_build: hg38
Supplementary_files_format_and_content: Bigwig files for each sample

source_name: 63.3 mouse GMP cells

extract_protocol:
Cells were stained with a final concentration of 1 ng/ml DAPI in growth media, and 1 million live cells were sorted using a BD FACSAria and placed on ice. CUT&RUN was then performed using the Epicypher CUTANA CUT&RUN kit, using the indicated number of cells, antibody, and concentration of antibody.
Library construction was performed using the NEBNext Ultra II Library Preparation Kit following the indicated protocol, and each library was indexed using NEBNext® Multiplex Oligos for Illumina. For PU.1, the library construction protocol was modified to generate shorter fragments per https://www.protocols.io/view/library-prep-for-cut-amp-run-with-nebnext-ultra-ii-wvgfe3w. Libraries were quantified using Qubit and Bioanalyzer and pooled for sequencing using an Illumina NextSeq 500.

value: Brequinar 2uM

value: H3K4me1, Millipore 07-436

tag: time point


title: 63.3_DMSO_IgG

description: CUT&RUN


title: 63.3_DMSO_H3K4me1

description: CUT&RUN


title: iPS201-RPE, 6 weeks, rep1, cell40


title: 63.3_BRQ_IgG

description: CUT&RUN


title: elder_5

type: RNA

city: Fukuoka

first: Takahiro

last: Tomiyama

state: Fukuoka

address: 3-1-1 Maidashi. Higashi-ku

department: Surgery and Science

institute: Kyushu University

description: Sample 12

hyb_protocol: The hybridization experiments were performed onto an Agilent SurePrint G3 M.fasciculari GE 8x60K Microarrays (G4858A#75606); this platform was designed from NCBI Refseq 2015 May Macaca fascicularis protein coding genes.

scan_protocol: The intensity of each hybridization signal was evaluated using SureScan Microarray Scanner (Agilent Technologies Inc.).

data_processing: Images were quantified using Agilent Feature Extraction Software (version 10.5.1.1).

organism: Macaca fascicularis

source_name: The liver tissue from elder (27 years old) cynomolgus macaques

label_protocol: Five hundred nanograms of total RNA was reverse-transcribed into double-stranded cDNA using AffinityScript multiple temperature reverse transcriptase (Agilent Technologies Inc.). The resulting complimentary RNAs were labeled with cyanine-3 (Perkin-Elmer) using a Low Input Quick-Amp Labeling kit (Agilent Technologies Inc.).

extract_protocol: RNA extraction, from cynomolgus macaque liver tissue, was performed, using the Maxwell® RSC RNA cells and RNA tissue kit (Promega Co., WI, USA).

tag: group

value: elder

tag: age

value: 27 years old

value: liver


title: iPS201-RPE, 6 weeks, rep1, cell45


title: 633_MULTIseq_run2_with_barcodes

description: scRNAseq


title: iPS201-RPE, 6 weeks, rep1, cell46


title: iPS201-RPE, 6 weeks, rep1, cell44


title: elder_3

description: Sample 10


title: iPS201-RPE, 6 weeks, rep1, cell36


title: ER-Hoxa9_dCas9KRAB_sgNTC2_DMSO_rep4


title: 63.3_BRQ_H3K4me3

description: CUT&RUN

value: H3K4me3, Epicypher 13-0028


title: iPS201-RPE, 6 weeks, rep1, cell42


title: elder_1

description: Sample 8

source_name: The liver tissue from elder (26 years old) cynomolgus macaques

value: 26 years old


title: iPS201-RPE, 6 weeks, rep1, cell35

value: iPSC derived RPE cell


title: 63.3_DMSO_H3K4me3

description: CUT&RUN


title: iPS201-RPE, 6 weeks, rep1, cell41

value: RPE


title: iPS201-RPE, 6 weeks, rep1, cell47


title: iPS201-RPE, 6 weeks, rep1, cell39


title: elder_6

description: Sample 13


title: young_5

description: Sample 5

source_name: The liver tissue from young (9 years old) cynomolgus macaques

value: young

value: 9 years old


title: young_6

description: Sample 6

source_name: The liver tissue from young (7 years old) cynomolgus macaques

value: 7 years old


title: elder_4

description: Sample 11


title: iPS201-RPE, 6 weeks, rep1, cell38

value: 6 weeks


title: iPS201-RPE, 6 weeks, rep1, cell37


title: iPS201-RPE, 6 weeks, rep1, cell31


title: young_4

description: Sample 4


title: HID1_ChIRP_input_Rep2

city: Beijing

first: Yangyang

last: Fan

address: No.5 Yiheyuan Road Haidian District,

country: China

institute: Peking University

description: input (negative control)

data_processing:
library strategy: ChIRP-seq
Illumina bcl-convert v3.7.5 software used for basecalling.
Raw ChIRP sequence reads were mapped to the TAIR10 genome assembly of Arabidopsis thaliana via bowtie2 (version 2.1.0) with default parameters.
Only uniquely mapped reads with high mapping quality (MAPQ > 30) were kept for further analysis.
Peak calling was performed using MACS (version 1.4.2) (Zhang, Liu et al. 2008) with the following parameters: --keep-dup 1 --gsize 1.2e8 --pvalue 1e5 --keep-dup=auto --to-large --nomodel --shiftsize 100.
Genome_build: TAIR10
Supplementary_files_format_and_content: Bigwig files were generated using bam2wig.py tool from RSeqQC.
Supplementary_files_format_and_content: peak text files

organism: Arabidopsis thaliana

source_name: Arabidopsis thaliana seeds

extract_protocol:
0.3 g seeds were used for each assay. The materials were fixed by two methods in the dark room. For reversible chemical cross-linking, fixing was performed with 1% (v/v) formaldehyde in PBS at room temperature for 20 min under vacuum. For non-reversible cross-linking, fixing was performed with 1% (v/v) glutaraldehyde in PBS at room temperature for 20 min under vacuum. For both cross-linking methods, the reactions were quenched with glycine (0.125 M final concentration) for 5 min under vacuum. Antisense DNA probes were designed against the full-length HID1 sequence and biotinylated at the 3′ end (Invitrogen). Unlabeled probes were used as the control. DNA was purified with the ChIP DNA Clean & Concentrator kit (D5205, Zymo research) according to the manufacturer’s instructions, and the purified DNA was subjected to high-throughput sequencing.
All ChIRP-seq libraries were generated using the NEBNext® Ultra II DNA Library Prep Kit for Illumina (E7645S, NEB) according to the manufacturer’s instructions.
All libraries were sequenced on a HiSeq 2500 System with paired-end 150 bp reads.

value: seeds

tag: developmental stage

value: seeds at 48 h of dark incubation after light treatment under the phyB-on condition

value: Col-0 ecotype


title: iPS201-RPE, 6 weeks, rep1, cell27

source_name: iPSC derived RPE cell


title: young_7

description: Sample 7

source_name: The liver tissue from young (8 years old) cynomolgus macaques

value: 8 years old


title: iPS201-RPE, 6 weeks, rep1, cell26


title: iPS201-RPE, 6 weeks, rep1, cell32


title: young_3

description: Sample 3


title: elder_2

description: Sample 9


title: iPS201-RPE, 6 weeks, rep1, cell30


title: iPS201-RPE, 6 weeks, rep1, cell29


title: young_1

description: Sample 1


title: HID1_ChIRP_input_Rep1

description: input (negative control)


title: HID1_ChIRP_mock_Rep2

description: Unlabeled probes were used as the control.


title: iPS201-RPE, 6 weeks, rep1, cell22


title: iPS201-RPE, 6 weeks, rep1, cell28


title: iPS201-RPE, 6 weeks, rep1, cell34


title: HID1_ChIRP_Rep1

description: For reversible chemical cross-linking, fixing was performed with 1% (v/v) formaldehyde in PBS at room temperature for 20 min under vacuum. Antisense DNA probes were designed against the full-length HID1 sequence and biotinylated at the 3′ end (Invitrogen).


title: iPS201-RPE, 6 weeks, rep1, cell24


title: ChIPseq_H3K27me3_48ON_phyB_rep2

description: H3K27me3-ChIP from phyB mutant rep2

data_processing:
Illumina bcl-convert v3.7.5 software used for basecalling.
After removing reads with adapter sequences and low quality by fastp, the high-quality reads were mapped to the TAIR10 genome assembly of Arabidopsis thaliana using bowtie2 (version 2.3.3) with default parameters.
Peak calling was performed using MACS2 (version 2.2.5) with the following parameters: --keep-dup 1 --gsize 1.2e8 --qvalue 0.05.
The alignments were converted to bigWig files using the bamCoverage tool from deepTools2 (version 3.1.3) with parameters “--binSize 5 --normalizeUsing CPM”.
Genome_build: TAIR10
Supplementary_files_format_and_content: Bigwig files were generated using the bamCoverage tool from deepTools2 (version 3.1.3) with parameters “--binSize 5 --normalizeUsing CPM”.
Supplementary_files_format_and_content: peak text files

extract_protocol:
0.3 g of imbibed seeds grown in the phyB-on condition for 48 h were collected and cross-linked with 1% formaldehyde for 20 min under vacuum and then ground into a fine powder in liquid nitrogen. Chromatin was isolated and sheared into fragments (200–500 bp) by sonication. The sonicated chromatin was incubated with antibody-bound Sera-Mag SpeedBeads Protein A/G Magnetic Particles (17152104010150, GE Healthcare) overnight at 4 ℃. Precipitated DNA was then reverse-crosslinked and digested with RNase A and Proteinase K, after which it was purified with the ChIP DNA Clean & Concentrator kit (D5205, Zymo research) according to the manufacturer’s instructions.
All ChIP-seq libraries were generated with the NEBNext® Ultra II DNA Library Prep Kit for Illumina® (E7645S, NEB) according to the manufacturer’s instructions.

tag: chip antibody

value: H3K27me3 (07-449, Millipore)

value: phyB mutant


title: iPS201-RPE, 6 weeks, rep1, cell19

tag: cell line


title: iPS201-RPE, 6 weeks, rep1, cell23


title: HID1_ChIRP_mock_Rep1


title: iPS201-RPE, 6 weeks, rep1, cell33


title: iPS201-RPE, 6 weeks, rep1, cell18


title: ChIPseq_H3K27me3_48ON_hid1_rep3

description: H3K27me3-ChIP from hid1 mutant rep3

value: hid1 mutant


title: iPS201-RPE, 6 weeks, rep1, cell20


title: ChIPseq_H3K27me3_48ON_phyB_rep3

description: H3K27me3-ChIP from phyB mutant rep3


title: HID1_ChIRP_Rep2

description: For non-reversible cross-linking, fixing was performed with 1% (v/v) glutaraldehyde in PBS at room temperature for 20 min under vacuum. Antisense DNA probes were designed against the full-length HID1 sequence and biotinylated at the 3′ end (Invitrogen).


title: iPS201-RPE, 6 weeks, rep1, cell21


title: ChIPseq_H3K27me3_48ON_phyB_rep1

description: H3K27me3-ChIP from phyB mutant rep1


title: young_2

description: Sample 2

source_name: The liver tissue from young (5 years old) cynomolgus macaques

value: 5 years old


title: iPS201-RPE, 6 weeks, rep1, cell14


title: iPS201-RPE, 6 weeks, rep1, cell16


title: ChIPseq_H3K27me3_48ON_hid1_rep2

description: H3K27me3-ChIP from hid1 mutant rep2


title: iPS201-RPE, 6 weeks, rep1, cell17


title: ChIPseq_H3K27me3_48ON_hid1_rep1

description: H3K27me3-ChIP from hid1 mutant rep1


title: iPS201-RPE, 6 weeks, rep1, cell12


title: ChIPseq_H3K27me3_48ON_WT_rep1

description: H3K27me3-ChIP from WT rep1


title: iPS201-RPE, 6 weeks, rep1, cell15


title: ChIPseq_H3K4me3_48ON_phyB_rep3

description: H3K4me3-ChIP from phyB mutant rep3

value: H3K4me3 (04-745, Millipore)


title: iPS201-RPE, 6 weeks, rep1, cell13


title: iPS201-RPE, 6 weeks, rep1, cell25


title: ChIPseq_H3K27me3_48ON_WT_rep2

description: H3K27me3-ChIP from WT rep2


title: iPS201-RPE, 6 weeks, rep1, cell11


title: ChIPseq_H3K27me3_48ON_WT_rep3

description: H3K27me3-ChIP from WT rep3


title: iPS201-RPE, 6 weeks, rep1, cell10


title: iPS201-RPE, 6 weeks, rep1, cell5


title: iPS201-RPE, 6 weeks, rep1, cell4


title: ChIPseq_H3K4me3_48ON_phyB_rep1

description: H3K4me3-ChIP from phyB mutant rep1


title: ChIPseq_H3K4me3_48ON_WT_rep3

description: H3K4me3-ChIP from WT rep3


title: ChIPseq_H3K4me3_48ON_WT_rep1

description: H3K4me3-ChIP from WT rep1


title: iPS201-RPE, 6 weeks, rep1, cell8


title: ChIPseq_H3K4me3_48ON_phyB_rep2

description: H3K4me3-ChIP from phyB mutant rep2


title: RNA-seq_48ON_phyB_rep3

data_processing:
Illumina bcl-convert v3.7.5 software used for basecalling.
After removing reads with adapter sequences and low quality by fastp, the high-quality reads were mapped to the TAIR10 genome assembly of Arabidopsis thaliana using HISAT2 (version 2.1.0) with parameters “--dta --min-intronlen 50 --max-intronlen 6000”. Only unique mapping and MAPQ score > 20 reads were kept using SAMtools version 1.6 for subsequent analysis.
Genome_build: TAIR10
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each sample
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample

extract_protocol:
Total RNA was isolated using the Spectrum Plant Total RNA Kit (STRN50, Sigma).  Three biological replicates for each sample were subjected to RNA-seq.
Poly(A) RNA from 1 μg total RNA was used to generate each cDNA library according to the TruSeq RNA Sample Prep Kit (Illumina) protocol. All libraries were then sequenced on a HiSeq 2500 System (Illumina) with paired-end 150-bp reads.


title: iPS201-RPE, 6 weeks, rep1, cell9


title: ChIPseq_H3K4me3_48ON_hid1_rep2

description: H3K4me3-ChIP from hid1 mutant rep2


title: ChIPseq_H3K4me3_48ON_hid1_rep3

description: H3K4me3-ChIP from hid1 mutant rep3


title: iPS201-RPE, 6 weeks, rep1, cell7


title: iPS201-RPE, 6 weeks, rep1, cell6


title: ChIPseq_H3K4me3_48ON_WT_rep2

description: H3K4me3-ChIP from WT rep2


title: RNA-seq_48ON_hid1_rep3


title: iPS201-RPE, 6 weeks, rep1, cell3


title: iPS201-RPE, 6 weeks, rep1, cell2


title: iPS253-RPE, 6 weeks, rep1, cell190


title: RNA-seq_48ON_hid1_rep1


title: RNA-seq_48ON_phyB_rep2


title: iPS253-RPE, 6 weeks, rep1, cell192


title: ChIPseq_H3K4me3_48ON_hid1_rep1

description: H3K4me3-ChIP from hid1 mutant rep1


title: RNA-seq_48ON_WT_rep2


title: iPS253-RPE, 6 weeks, rep1, cell191


title: RNA-seq_48ON_hid1_rep2


title: RNA-seq_24ON_phyB_rep2

value: seeds at 24 h of dark incubation after light treatment under the phyB-on condition


title: iPS253-RPE, 6 weeks, rep1, cell188


title: RNA-seq_48ON_WT_rep3


title: RNA-seq_48ON_phyB_rep1


title: iPS253-RPE, 6 weeks, rep1, cell185


title: iPS253-RPE, 6 weeks, rep1, cell186


title: RNA-seq_24ON_hid1_rep3


title: iPS201-RPE, 6 weeks, rep1, cell1


title: RNA-seq_24ON_phyB_rep1


title: iPS253-RPE, 6 weeks, rep1, cell189


title: iPS253-RPE, 6 weeks, rep1, cell187


title: RNA-seq_24ON_hid1_rep1


title: RNA-seq_24ON_hid1_rep2


title: iPS253-RPE, 6 weeks, rep1, cell184


title: iPS253-RPE, 6 weeks, rep1, cell180


title: iPS253-RPE, 6 weeks, rep1, cell183


title: RNA-seq_24ON_WT_rep3


title: RNA-seq_24ON_WT_rep2


title: iPS253-RPE, 6 weeks, rep1, cell182


title: iPS253-RPE, 6 weeks, rep1, cell177


title: RNA-seq_48ON_WT_rep1


title: iPS253-RPE, 6 weeks, rep1, cell179


title: iPS253-RPE, 6 weeks, rep1, cell178


title: RNA-seq_24ON_WT_rep1


title: RNA-seq_12ON_phyB_rep2

value: seeds at 12 h of dark incubation after light treatment under the phyB-on condition


title: RNA-seq_12ON_phyB_rep3


title: RNA-seq_24ON_phyB_rep3


title: iPS253-RPE, 6 weeks, rep1, cell176

value: iPSC derived RPE cell


title: iPS253-RPE, 6 weeks, rep1, cell174

value: RPE


title: iPS253-RPE, 6 weeks, rep1, cell181


title: RNA-seq_12ON_hid1_rep3


title: iPS253-RPE, 6 weeks, rep1, cell173


title: iPS253-RPE, 6 weeks, rep1, cell172


title: RNA-seq_12ON_WT_rep3


title: RNA-seq_12ON_WT_rep2


title: iPS253-RPE, 6 weeks, rep1, cell170


title: iPS253-RPE, 6 weeks, rep1, cell169


title: iPS253-RPE, 6 weeks, rep1, cell171


title: RNA-seq_12ON_WT_rep1


title: RNA-seq_12ON_hid1_rep1


title: iPS253-RPE, 6 weeks, rep1, cell168


title: iPS253-RPE, 6 weeks, rep1, cell166


title: 633_E2wd120_rep1_ATACseq

description: ATACseq

data_processing:
Mouse and human data were trimmed using Cutadapt (v3.4) (with parameters -m 20 -q 20 -a CTGTCTCTTATA -A CTGTCTCTTATA), then aligned to the mm10 and hg38 genomes with Bowtie2 v2.4.2 (with parameters --dovetail -I 10 -X 1000 --very-sensitive).
Reads in the mitochondrial genome and unannotated chromosomes as well as the UCSC blacklist were removed, then duplicates were removed with samtools markdup (v1.1.2), and the insert size distribution was calculated using Picard CollectInsertSizeMetrics (v2.25.2) and validated to contain nucleosomal peaks. BED file was then constructed with each mate pair as a separate read using bedtools bamtobed (v2.3.0), and peaks were called using MACS2 callpeak (v2.2.7.1) (with parameters --nomodel --shift -100 --extsize 200 --keep-dup all --call-summits). Note that the MACS2 command shifts each read by 100 base pairs towards the 5’ end and extends the read length to 200 base pairs, thus treating the 5’ end of the original read (the Tn5 cut site) as the center of a new “fragment” that is then used for peak calling and other downstream analysis.
To normalize tracks, reads overlapping shared peaks were counted for each BAM file using featureCounts (with parameters -M -O) and normalization factors were calculated from these counts using the calcNormFactors function in the edgeR package (v3.32.1) in R. Visualization was performed using the bedGraphToBigWig utility and the igv-jupyter package in Python.
Genome_build: hg38
Genome_build: mm10
Supplementary_files_format_and_content: Bigwig files for each sample

growth_protocol: Cells were grown in RPMI (Corning, 15-040-CV) with 10% dialyzed fetal bovine serum (Sigma, F0392), 4mM glutamine, and 1% penicillin/streptomycin (Corning, 45000-652), supplemented with 0.5uM beta-estradiol (Sigma, E2758; made from a 10mM stock dissolved in 100% ethanol) and stem cell factor (SCF) from conditioned media generated from a Chinese hamster ovary (CHO) cell line that stably secretes SCF (Sykes et al. Cell 2016; this conditioned media was added at a concentration of 1%). For treatment, cells were washed 2x in PBS and then resuspended in the identical media as above without estrogen.

tag: treatment

value: Estrogen withdrawal


title: 633_E2wd96_rep2_ATACseq

description: ATACseq


title: iPS253-RPE, 6 weeks, rep1, cell167

tag: tissue


title: 633_E2wd96_rep1_ATACseq

description: ATACseq


title: iPS253-RPE, 6 weeks, rep1, cell165

tag: cell type


title: iPS253-RPE, 6 weeks, rep1, cell164

tag: rpe culture_period


title: iPS253-RPE, 6 weeks, rep1, cell175


title: 633_E2wd72_rep2_ATACseq

description: ATACseq


title: 633_E2wd72_rep1_ATACseq

description: ATACseq


title: RNA-seq_12ON_phyB_rep1

tag: genotype


title: iPS253-RPE, 6 weeks, rep1, cell162


title: 633_E2wd48_rep2_ATACseq

description: ATACseq


title: 633_E2wd36_rep1_ATACseq

description: ATACseq


title: 633_E2wd120_rep2_ATACseq

description: ATACseq


title: 633_E2wd48_rep1_ATACseq

description: ATACseq


title: iPS253-RPE, 6 weeks, rep1, cell161


title: iPS253-RPE, 6 weeks, rep1, cell157


title: iPS253-RPE, 6 weeks, rep1, cell159


title: RNA-seq_12ON_hid1_rep2


title: iPS253-RPE, 6 weeks, rep1, cell160


title: 633_E2wd36_rep2_ATACseq

description: ATACseq


title: iPS253-RPE, 6 weeks, rep1, cell153


title: 633_E2wd24_rep2_ATACseq

description: ATACseq


title: iPS253-RPE, 6 weeks, rep1, cell156


title: iPS253-RPE, 6 weeks, rep1, cell155


title: 633_E2wd24_rep1_ATACseq

description: ATACseq


title: 633_E2wd12_rep2_ATACseq

description: ATACseq


title: 633_E2wd12_rep1_ATACseq

description: ATACseq


title: 633_E2wd4_rep1_ATACseq

description: ATACseq


title: 633_E2wd0_rep2_ATACseq

description: ATACseq


title: iPS253-RPE, 6 weeks, rep1, cell154


title: 633_E2wd8_rep2_ATACseq

description: ATACseq


title: 633_E2wd8_rep1_ATACseq

description: ATACseq


title: iPS253-RPE, 6 weeks, rep1, cell163


title: 633_E2wd4_rep2_ATACseq

description: ATACseq


title: iPS253-RPE, 6 weeks, rep1, cell152


title: 633_E2wd0_rep1_ATACseq

description: ATACseq


title: iPS253-RPE, 6 weeks, rep1, cell158


title: iPS253-RPE, 6 weeks, rep1, cell151


title: iPS253-RPE, 6 weeks, rep1, cell150


title: iPS253-RPE, 6 weeks, rep1, cell147


title: iPS253-RPE, 6 weeks, rep1, cell149


title: iPS253-RPE, 6 weeks, rep1, cell144


title: 633_BRQ36_rep1_ATACseq

description: ATACseq

value: Brequinar 2uM


title: iPS253-RPE, 6 weeks, rep1, cell146


title: iPS253-RPE, 6 weeks, rep1, cell148


title: 633_BRQ48_rep2_ATACseq

description: ATACseq


title: 633_BRQ36_rep2_ATACseq

description: ATACseq


title: 633_BRQ48_rep1_ATACseq

description: ATACseq


title: iPS253-RPE, 6 weeks, rep1, cell145


title: iPS253-RPE, 6 weeks, rep1, cell143


title: iPS253-RPE, 6 weeks, rep1, cell141


title: 633_BRQ24_rep1_ATACseq

description: ATACseq


title: 633_BRQ24_rep2_ATACseq

description: ATACseq


title: 633_BRQ12_rep2_ATACseq

description: ATACseq


title: iPS253-RPE, 6 weeks, rep1, cell142


title: iPS253-RPE, 6 weeks, rep1, cell140


title: 633_BRQ4_rep2_ATACseq

description: ATACseq


title: iPS253-RPE, 6 weeks, rep1, cell134


title: 633_BRQ12_rep1_ATACseq

description: ATACseq


title: iPS253-RPE, 6 weeks, rep1, cell138


title: iPS253-RPE, 6 weeks, rep1, cell135


title: 633_BRQ0_rep1_ATACseq

description: ATACseq


title: iPS253-RPE, 6 weeks, rep1, cell137


title: iPS253-RPE, 6 weeks, rep1, cell136


title: iPS253-RPE, 6 weeks, rep1, cell133


title: iPS253-RPE, 6 weeks, rep1, cell132


title: 633_BRQ8_rep2_ATACseq

description: ATACseq


title: iPS253-RPE, 6 weeks, rep1, cell130


title: THP1PIP_H3K27ac_HU100_8h_Early_r1

description: Sorted, early S, treated for 8 hours, not on ice prior to CUT&RUN

data_processing:
Mouse and human data were mapped to mm10 and hg38 genomes, respectively, using Bowtie2 (with parameters --dovetail -I 10 -X 700 --very-sensitive-local --local), then normalized using the bamCoverage tool from deepTools (with parameters -bs 1 -e 200). Duplication rates were assessed using Picard, and as they were below 15% for all samples, duplicate reads were not removed.
For H3K27ac CUT&RUN, read counts were tabulated for each peak in the master ATAC-seq peak list, and differential peaks were identified using edgeR. Motif enrichment at sites of differential H3K27ac signal was determined using HOMER. Peaks corresponding to promoter sites were identified by filtering out peaks that overlapped a +/-2kb region around promoters defined in Ensembl (org.Mm.eg.db or org.Hs.eg.db), using the package ChIPseeker. All other peaks were considered to be distal regulatory loci, and they were linked to genes using GREAT using the “single nearest gene” rule; associations were retained if the center of the peak was within 100kb of the nearest gene’s TSS. For the early S phase CUT&RUN (Figure S7F-J), H3K27ac peaks were defined using MACS2 with parameters “-q 0.01 --nomodel”, and the union of peaks that appeared in both replicates of each condition was used as the master peak list. For ER-Hoxa9 CUT&RUN, peaks were defined using MACS2 using default parameters. For RNA polymerase II CUT&RUN, gene bodies were defined as the region from position +250 (downstream of the promoter) to the transcription termination site (TTS). Heatmaps and metaplots were generated using deepTools.
Assembly: hg38, mm10
Supplementary files format and content: bigwig
Library strategy: CUT&RUN

source_name: THP-1 PIP-FUCCI

growth_protocol: Cells were grown in RPMI (Corning, 15-040-CV) with 10% dialyzed fetal bovine serum (Sigma, F0392), 4mM glutamine, and 1% penicillin/streptomycin (Corning, 45000-652). For ER-Hoxa9 cells, media was supplemented with 0.5uM beta-estradiol (Sigma, E2758; made from a 10mM stock dissolved in 100% ethanol) and stem cell factor (SCF) from conditioned media generated from a Chinese hamster ovary (CHO) cell line that stably secretes SCF (Sykes et al. Cell 2016; this conditioned media was added at a concentration of 1%).

extract_protocol:
CUT&RUN was performed using the Epicypher CUTANA CUT&RUN kit, using the indicated concentration of antibody.
Library construction was performed using the NEBNext Ultra II Library Preparation Kit following the indicated protocol, and each library was indexed using NEBNext® Multiplex Oligos for Illumina. Libraries were quantified using Qubit and Bioanalyzer and pooled for sequencing using an Illumina NextSeq 500.

treatment_protocol: For early S phase cell isolation experiments, 40 million THP-1 wild-type cells or cells expressing the PIP-FUCCI system were concentrated to a final concentration of 2 million/ml. DyeCycle Violet was added to a final concentration of 5 µM, and the cells were mixed well and returned to the incubator for 1 hour. At the 40-minute timepoint, ¼ of the cells was spiked with 20 µM EdU and returned to the incubator. At the 1-hour timepoint, cells were concentrated to 10 million/ml, the pellet was resuspended in the appropriate amount of supernatant containing DyeCycle Violet, and 500 µl of cells was aliquoted into flow cytometry tubes and placed on ice and sorted. S phase cells were identified as those that were Cdt1-mVenuslow, and S phase cells in the bottom 1/3 of DyeCycle Violet signal (a proxy for DNA content) were considered to be in early S phase. Cells were sorted in a chamber cooled to 4C and kept on ice prior to HU treatment or immediate CUT&RUN processing in order to minimize further cell cycle progression. Sorting was performed on BD FACSAria machines at the Koch Institute flow cytometry core, using the 70 μm nozzle. All cells were sorted into fresh cell culture media and kept on ice unless otherwise indicated.

value: THP-1 PIP-FUCCI

value: H3K27ac, Millipore MABE647

value: HU 100 uM 8h


title: Erythroid_MULTIseq_cell_barcodes

description: barcode library

library_source: transcriptomic single cell

data_processing:
For data analysis of human CD34-derived erythroid progenitors, FASTQ files were mapped to the human cDNA transcriptome with the 10x v3 whitelist (files available at https://github.com/pachterlab/kallisto-transcriptome-indices/releases) using Kallisto. Samples were then demultiplexed using the deMULTIplex package in R and doublets were discarded, then processed using Seurat.
Genes expressed in fewer than 3 cells or cells with fewer than 200 distinct transcripts or with a mtRNA percentage of >10% were discarded.
Assembly: hg38
Supplementary files format and content: Cell Ranger/Kallisto mtx file for gene counts for each cell along with corresponding barcodes.txt and genes.txt annotation files, as well as a multi_seq_cell_classifications.csv file for assigning each cell to a sample barcode and multiseq_barcodes.txt which associates sample barcodes with sample name (donor ID, day of harvest, and treatment).

source_name: CD34 cells differentiated down erythroid lineage

growth_protocol: For primary human CD34+ HSPCs, cells were obtained from the Fred Hutchinson Hematopoietic Cell Procurement and Resource Development repository. No human subjects were involved in this study. Each vial had greater than 90% CD34 purity and had been positively selected using the Miltenyi CliniMacs system. Donors were deidentified and were negative for standard virology screening. Cells were differentiated into mature erythroid cells using a three-phase culture protocol, with base media consisting of Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 2% human blood plasma, 3% human AB serum, 3 U/ml heparin, and 10 µg/ml insulin. In phase 1 (day 0-7), cells were cultured at a density of 1e5-1e6/ml in base media supplemented with 200 µg/ml holo-transferrin, 10 ng/ml SCF, 1 ng/ml IL-3, and 3U/ml Epo. In phase 2 (days 7-11), cells were cultured at a density of 1e5-1e6/ml in base media supplemented with 200 µg/ml holo-transferrin, 10 ng/ml SCF, and 3U/ml Epo. In phase 3 (days 11-15), cells were cultured at a density of 1e6-5e6/ml in base media supplemented with 500 µg/ml holo-transferrin and 0.1U/ml Epo.

extract_protocol:
Briefly, for each condition, 400,000 cells were rinsed twice in PBS and resuspended in 180 μl PBS. 20 μl of a 10X stock of anchor:barcode (per sample: 0.88 μl anchor, 0.44 μl 100 μM barcode, and 20.7 μl PBS) was added to each sample, pipetted gently to mix, and incubated on ice for 5 minutes. Next, 20 μl of a 10X stock of coanchor (per sample: 0.88 μl co-anchor and 21.2 μl PBS) was added to each sample, pipetted gently to mix, and incubated on ice for 5 minutes. 700 μl ice cold 1% BSA in PBS was then added to each sample, and cells were centrifuged at 4ºC for 5 minutes. The supernatant was aspirated and the wash was repeated 2X with ice cold BSA in PBS. After the last wash, cells were resuspended in 300 μl PBS/BSA, filtered through a cell strainer, and counted using a Nexcelom cell counter. Cells were adjusted to a concentration of 1 million/ml using ice cold BSA in PBS, and 50 μl of each sample was pooled into one tube and placed on ice. Cells were then loaded into the 10X Genomics microfluidics chip for processing and scRNAseq. For the ER-Hoxa9 experiment, 28,500 cells were loaded in order to obtain enough data for ~15,000 usable cells, and for the erythroid progenitor experiment, 22,000 cells were loaded in order to obtain enough data for ~12,000 usable cells (calculation performed at https://satijalab.org/costpercell/).
Library construction proceeded as indicated in the 10X Genomics scRNAseq v3 protocol except as follows (adapted from McGinnis et al.). During cDNA amplification, 1 μl of a 2.5 μM MULTI-seq primer was added to the master mix. After the 0.6X SPRI clean-up, the supernatant was saved for barcode extraction rather than discarded. To the 60 μl supernatant, 260 μl SPRI beads and 180 μl 100% isopropanol were added for a final ratio of 1.8X beads, pipetted up and down 10x, and incubated at room temperature for 5 minutes. The tube was then placed on a magnetic rack for 5 minutes, supernatant discarded, beads washed 2x with 500 μl 80% ethanol, and eluted with 50 μl buffer EB. This SPRI purification step was repeated once more to further purify the barcode fraction. The barcode fraction was quantified using Qubit, and then library preparation PCR was performed using the Kapa HiFi HotStart ReadyMix with 3.5 ng barcode DNA, 2.5 μl 10 μM universal i5 primer, and 2.5 μl 10 μM TruSeq RPI1 primer in a 50 μl reaction for 8-12 cycles. The PCR product was purified using a final SPRI bead concentration of 1.6X, eluted, and quantified on a Bioanalyzer; a peak between 175 and 200bp was expected. The barcode library was mixed 1:10 with the transcript library and sequenced on an Illumina NextSeq 500.

treatment_protocol: CD34+ HSPCs differentiated down the erythroid lineage as described above were treated with DMSO, 50 µM HU, or 500 nM APH starting at day 7; 1 million cells from each sample were frozen down in 90% FBS and 10% DMSO and cryopreserved at day 11 and 14 of the differentiation protocol. On the day of MULTI-seq processing, vials were thawed per standard CD34 protocols, then were barcoded and pooled.


title: iPS253-RPE, 6 weeks, rep1, cell131

value: 6 weeks


title: THP1PIP_H3K27ac_Unt_Early_r2

description: Sorted, early S, on ice prior to CUT&RUN


title: THP1PIP_H3K27ac_HU100_8h_Early_r2


title: THP1PIP_H3K27ac_Unt_Early_r1


title: 633_BRQ8_rep1_ATACseq

description: ATACseq


title: iPS253-RPE, 6 weeks, rep1, cell139


title: 633_BRQ4_rep1_ATACseq

description: ATACseq


title: iPS253-RPE, 6 weeks, rep1, cell129


title: iPS253-RPE, 6 weeks, rep1, cell128


title: THP1_H3K27ac_Unt_Unsorted_r2

description: Unsorted, not on ice prior to CUT&RUN


title: iPS253-RPE, 6 weeks, rep1, cell126


title: 633_BRQ0_rep2_ATACseq

description: ATACseq


title: Erythroid_MULTIseq_transcripts

description: transcript library

value: CD34 cells differentiated down erythroid lineage


title: iPS253-RPE, 6 weeks, rep1, cell127


title: THP1_H3K27ac_Unt_SortedAll_r2

description: Sorted, on ice prior to CUT&RUN


title: iPS253-RPE, 6 weeks, rep1, cell125


title: THP1_H3K27ac_Unt_Unsorted_r1

description: Unsorted, not on ice prior to CUT&RUN


title: iPS253-RPE, 6 weeks, rep1, cell122


title: iPS253-RPE, 6 weeks, rep1, cell123


title: THP1_PIP_EarlyS_8h_r1

data_processing:
Mouse and human data were mapped to mm10 and hg38 genomes, respectively, using STAR and default parameters. Reads that mapped to transcripts were counted using featureCounts (with parameters -M -O --fraction --primary). Normalization and differential gene expression analysis was performed using the edgeR package in R.
Assembly: hg38
Supplementary files format and content: csv; raw counts

extract_protocol:
Immediately following the sort, cells were centrifuged, pelleted, and flash frozen in liquid nitrogen and placed at -80ºC. Pellets were processed in groups of 24 using the Zymo RNA Miniprep Kit and quantified using the Invitrogen Quant-it RNA kit.
For THP-1 PIP-FUCCI experiments, 100 ng RNA was subjected to poly-A enrichment, cDNA synthesis, and library prep using the Ultra II Directional RNA-Seq Kit and reads were sequenced 2x75bp on an Illumina NextSeq 500.

treatment_protocol: For early S phase cell isolation experiments, 40 million THP-1 cells expressing the PIP-FUCCI system were concentrated to a final concentration of 2 million/ml. DyeCycle Violet was added to a final concentration of 5 µM, and the cells were mixed well and returned to the incubator for 1 hour. At the 40-minute timepoint, ¼ of the cells was spiked with 20 µM EdU and returned to the incubator. At the 1-hour timepoint, cells were concentrated to 10 million/ml, the pellet was resuspended in the appropriate amount of supernatant containing DyeCycle Violet, and 500 µl of cells was aliquoted into flow cytometry tubes and placed on ice and sorted. S phase cells were identified as those that were Cdt1-mVenuslow, and S phase cells in the bottom 1/3 of DyeCycle Violet signal (a proxy for DNA content) were considered to be in early S phase. Cells were sorted in a chamber cooled to 4C and kept on ice prior to HU treatment or immediate RNAseq/CUT&RUN processing in order to minimize further cell cycle progression. To validate our sorting approach, EdU-treated cells were sorted in an identical manner and fixed immediately after sorting, then subjected to processing with the Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Assay Kit and stained with DAPI. Similarly, a small aliquot of early S phase cells treated with HU for 8 hours was also spiked with EdU for 20 minutes, fixed, and processed for EdU/DAPI flow cytometry analysis. Sorting was performed on BD FACSAria machines at the Koch Institute flow cytometry core, using the 70 μm nozzle. All cells were sorted into fresh cell culture media and kept on ice unless otherwise indicated.

value: PIP-FUCCI


title: iPS253-RPE, 6 weeks, rep1, cell124


title: THP1_PIP_EarlyS_8h_r2


title: THP1_PIP_EarlyS_0h_r2


title: iPS253-RPE, 6 weeks, rep1, cell119


title: THP1_PIP_EarlyS_0h_r1


title: iPS253-RPE, 6 weeks, rep1, cell121


title: Sublibrary_THP-1_DMSO_CD14_Low

source_name: THP-1 dCas9-KRAB cells

value: THP-1 dCas9-KRAB cells

value: DMSO


title: huCRISPRi_library1_pcr

source_name: Addgene 83969

value: Addgene 83969


title: iPS253-RPE, 6 weeks, rep1, cell120


title: huCRISPRi_library2_pcr

source_name: Addgene 83970

value: Addgene 83970


title: iPS253-RPE, 6 weeks, rep1, cell117


title: iPS253-RPE, 6 weeks, rep1, cell118


title: THP1_H3K27ac_Unt_SortedAll_r1

description: Sorted, on ice prior to CUT&RUN


title: Sublibrary_THP-1_DMSO_CD11b_Low


title: iPS253-RPE, 6 weeks, rep1, cell116


title: Sublibrary_THP-1_DMSO_CD14_High


title: Sublibrary_THP-1_DMSO_CD11b_High


title: iPS253-RPE, 6 weeks, rep1, cell115


title: Sublibrary_THP-1_BRQ_CD14_Low

value: Brequinar 500nM


title: iPS253-RPE, 6 weeks, rep1, cell113


title: iPS253-RPE, 6 weeks, rep1, cell114


title: Sublibrary_THP-1_BRQ_CD14_High


title: iPS253-RPE, 6 weeks, rep1, cell112


title: iPS253-RPE, 6 weeks, rep1, cell111


title: Sublibrary_THP-1_BRQ_CD11b_High


title: Sublibrary_K562_DMSO_CD235a_Low

source_name: K562 dCas9-KRAB cells

value: K562 dCas9-KRAB cells


title: K562_i4_sgGATA1-1_BRQ_72_rep3

source_name: K562 dCas9-KRAB human CML cells (sgGATA1-1)

value: Brequinar 250nM


title: iPS253-RPE, 6 weeks, rep1, cell109

value: iPSC derived RPE cell


title: Sublibrary_THP-1_BRQ_CD11b_Low


title: Sublibrary_K562_DMSO_CD235a_High


title: iPS253-RPE, 6 weeks, rep1, cell110

value: RPE


title: iPS253-RPE, 6 weeks, rep1, cell108


title: K562_i4_sgGATA1-1_BRQ_72_rep2


title: iPS253-RPE, 6 weeks, rep1, cell104


title: K562_i4_sgGATA1-1_DMSO_72_rep3


title: iPS253-RPE, 6 weeks, rep1, cell107


title: K562_i4_sgGATA1-1_DMSO_72_rep2


title: iPS253-RPE, 6 weeks, rep1, cell106


title: K562_i4_sgGATA1-1_BRQ_72_rep1


title: iPS253-RPE, 6 weeks, rep1, cell105


title: iPS253-RPE, 6 weeks, rep1, cell103


title: K562_i4_sgGATA1-1_DMSO_72_rep1


title: iPS253-RPE, 6 weeks, rep1, cell102


title: iPS253-RPE, 6 weeks, rep1, cell100


title: K562_i4_sgNTC1_BRQ_72_rep1

source_name: K562 dCas9-KRAB human CML cells (sgNTC-1)


title: K562_i4_sgNTC1_BRQ_72_rep3


title: K562_i4_sgNTC1_BRQ_72_rep2


title: iPS253-RPE, 6 weeks, rep1, cell101


title: K562_i4_sgNTC1_DMSO_72_rep2


title: iPS253-RPE, 6 weeks, rep1, cell98


title: iPS253-RPE, 6 weeks, rep1, cell99


title: THP-1_HU_1_8_rep4

source_name: THP1 human AML cells

value: Hydroxyurea 100uM


title: K562_i4_sgNTC1_DMSO_72_rep3


title: THP-1_HU_1_8_rep3


title: iPS253-RPE, 6 weeks, rep1, cell97


title: iPS253-RPE, 6 weeks, rep1, cell93


title: iPS253-RPE, 6 weeks, rep1, cell94


title: THP-1_HU_1_8_rep2


title: iPS253-RPE, 6 weeks, rep1, cell95


title: THP-1_HU_1_4_rep4


title: iPS253-RPE, 6 weeks, rep1, cell91


title: THP-1_HU_1_8_rep1


title: THP-1_HU_1_4_rep1


title: THP-1_BRQ_500_24_rep4


title: THP-1_HU_1_4_rep3


title: iPS253-RPE, 6 weeks, rep1, cell96


title: iPS253-RPE, 6 weeks, rep1, cell86


title: THP-1_HU_1_4_rep2


title: iPS253-RPE, 6 weeks, rep1, cell87


title: THP-1_BRQ_500_24_rep3


title: iPS253-RPE, 6 weeks, rep1, cell89


title: THP-1_BRQ_500_24_rep2


title: K562_i4_sgNTC1_DMSO_72_rep1


title: iPS253-RPE, 6 weeks, rep1, cell84


title: THP-1_BRQ_500_8_rep4


title: THP-1_BRQ_500_24_rep1


title: iPS253-RPE, 6 weeks, rep1, cell92


title: THP-1_BRQ_500_8_rep1


title: THP-1_BRQ_500_8_rep3


title: iPS253-RPE, 6 weeks, rep1, cell83


title: iPS253-RPE, 6 weeks, rep1, cell80


title: iPS253-RPE, 6 weeks, rep1, cell79


title: iPS253-RPE, 6 weeks, rep1, cell82


title: iPS253-RPE, 6 weeks, rep1, cell88


title: THP-1_BRQ_500_8_rep2


title: iPS253-RPE, 6 weeks, rep1, cell78


title: THP-1_BRQ_500_4_rep2


title: iPS253-RPE, 6 weeks, rep1, cell81


title: THP-1_BRQ_500_4_rep3


title: iPS253-RPE, 6 weeks, rep1, cell77


title: iPS253-RPE, 6 weeks, rep1, cell90


title: iPS253-RPE, 6 weeks, rep1, cell85


title: iPS253-RPE, 6 weeks, rep1, cell76


title: THP-1_DMSO_0_rep2


title: iPS253-RPE, 6 weeks, rep1, cell73


title: iPS253-RPE, 6 weeks, rep1, cell74


title: THP-1_BRQ_500_4_rep1


title: THP-1_DMSO_0_rep4


title: THP-1_PMA_48_rep4

value: PMA 100nM


title: iPS253-RPE, 6 weeks, rep1, cell70


title: THP-1_BRQ_500_4_rep4


title: THP-1_PMA_48_rep3


title: iPS253-RPE, 6 weeks, rep1, cell68


title: THP-1_DMSO_0_rep3


title: iPS253-RPE, 6 weeks, rep1, cell75


title: THP-1_PMA_48_rep2


title: THP-1_DMSO_0_rep1


title: iPS253-RPE, 6 weeks, rep1, cell66


title: iPS253-RPE, 6 weeks, rep1, cell67


title: THP-1_BRQ_48_rep2


title: iPS253-RPE, 6 weeks, rep1, cell69


title: iPS253-RPE, 6 weeks, rep1, cell72


title: THP-1_PMA_48_rep1


title: iPS253-RPE, 6 weeks, rep1, cell64


title: THP-1_BRQ_48_rep3


title: THP-1_PMA_24_rep3


title: iPS253-RPE, 6 weeks, rep1, cell65


title: THP-1_BRQ_48_rep1


title: iPS253-RPE, 6 weeks, rep1, cell71


title: THP-1_PMA_24_rep1


title: iPS253-RPE, 6 weeks, rep1, cell62


title: iPS253-RPE, 6 weeks, rep1, cell61


title: THP-1_PMA_24_rep2


title: iPS253-RPE, 6 weeks, rep1, cell63


title: iPS253-RPE, 6 weeks, rep1, cell60


title: THP-1_BRQ_24_rep4


title: THP-1_BRQ_48_rep4


title: iPS253-RPE, 6 weeks, rep1, cell58


title: THP-1_BRQ_24_rep2


title: iPS253-RPE, 6 weeks, rep1, cell59


title: THP-1_BRQ_24_rep3


title: THP-1_BRQ_24_rep1


title: iPS253-RPE, 6 weeks, rep1, cell56


title: THP-1_PMA_24_rep4


title: iPS253-RPE, 6 weeks, rep1, cell55


title: THP-1_DMSO_48_rep2


title: iPS253-RPE, 6 weeks, rep1, cell54


title: iPS253-RPE, 6 weeks, rep1, cell53


title: THP-1_DMSO_48_rep1


title: THP-1_DMSO_48_rep3


title: THP-1_sgABCB7-1_rep3

source_name: THP1 human AML cells (dCas9-Zim3-KRAB)


title: ER-Hoxa9_BRQ_96h_rep2

description:
RNAseq
BRQ_96h_rep2

data_processing:
Mouse and human data were mapped to mm10 and hg38 genomes, respectively, using STAR v2.7.8a and default parameters.
Reads that mapped to transcripts were counted using featureCounts v2.0.1 (with parameters -M -O --fraction --primary).
Genome_build: mm10
Genome_build: hg38
Supplementary_files_format_and_content: A tab-delimited file containing raw counts generated from featureCounts.

source_name: 63.3 mouse GMP cells

extract_protocol:
Qiagen RNEasy PLUS kit with additional on-column DNAse step. Total RNA is quantified using the Quant-iT™ RiboGreen® RNA Assay Kit and normalized to 5ng/ul. An aliquot of 200ng for each sample is transferred into library preparation which is an automated variant of the Illumina TruSeq™ Stranded mRNA Sample Preparation Kit. This method preserves strand orientation of the RNA transcript. It uses oligo dT beads to select mRNA from the total RNA sample. It is followed by heat fragmentation and cDNA synthesis from the RNA template. The resultant 500bp cDNA then goes through library preparation (end repair, base ‘A’ addition, adapter ligation, and enrichment) using Broad designed indexed adapters substituted in for multiplexing. After enrichment the libraries were quantified with qPCR using the KAPA Library Quantification Kit for Illumina Sequencing Platforms and then pooled equimolarly. The entire process is in 96-well format and all pipetting is done by either Agilent Bravo or Hamilton Starlet. Pooled libraries are normalized to 2nM and denatured using 0.1 N NaOH prior to sequencing. Flowcell cluster amplification and sequencing were performed according to the manufacturer’s protocols using either the HiSeq 2000 or HiSeq 2500. Each run is a 101bp paired-end with an eight-base index barcode read. Data is analyzed using the Broad Picard Pipeline which includes de-multiplexing and data aggregation.
An aliquot of 200ng for each sample is transferred into library preparation which is an automated variant of the Illumina TruSeq™ Stranded mRNA Sample Preparation Kit. This method preserves strand orientation of the RNA transcript. It uses oligo dT beads to select mRNA from the total RNA sample. It is followed by heat fragmentation and cDNA synthesis from the RNA template. The resultant 500bp cDNA then goes through library preparation (end repair, base ‘A’ addition, adapter ligation, and enrichment) using Broad designed indexed adapters substituted in for multiplexing. After enrichment the libraries were quantified with qPCR using the KAPA Library Quantification Kit for Illumina Sequencing Platforms and then pooled equimolarly. The entire process is in 96-well format and all pipetting is done by either Agilent Bravo or Hamilton Starlet. Pooled libraries are normalized to 2nM and denatured using 0.1 N NaOH prior to sequencing. Flowcell cluster amplification and sequencing were performed according to the manufacturer’s protocols using either the HiSeq 2000 or HiSeq 2500. Each run is a 101bp paired-end with an eight-base index barcode read. Data is analyzed using the Broad Picard Pipeline which includes de-multiplexing and data aggregation.

value: Brequinar 2uM


title: iPS253-RPE, 6 weeks, rep1, cell51


title: ER-Hoxa9_BRQ_72h_rep2

description:
RNAseq
BRQ_72h_rep2


title: THP-1_sgABCB7-1_rep2


title: iPS253-RPE, 6 weeks, rep1, cell50


title: ER-Hoxa9_BRQ_72h_rep1

description:
RNAseq
BRQ_72h_rep1


title: iPS253-RPE, 6 weeks, rep1, cell49


title: THP-1_sgABCB7-1_rep1


title: iPS253-RPE, 6 weeks, rep1, cell57


title: ER-Hoxa9_BRQ_48h_rep2

description:
RNAseq
BRQ_48h_rep2


title: ER-Hoxa9_BRQ_48h_rep1

description:
RNAseq
BRQ_48h_rep1


title: iPS253-RPE, 6 weeks, rep1, cell48


title: THP-1_sgPOLD3-2_rep1


title: THP-1_DMSO_48_rep4


title: ER-Hoxa9_BRQ_24h_rep2

description:
RNAseq
BRQ_24h_rep2


title: ER-Hoxa9_BRQ_12h_rep2

description:
RNAseq
BRQ_12h_rep2


title: THP-1_sgPOLD3-2_rep2


title: ER-Hoxa9_BRQ_24h_rep1

description:
RNAseq
BRQ_24h_rep1


title: ER-Hoxa9_BRQ_8h_rep1

description:
RNAseq
BRQ_8h_rep1


title: iPS253-RPE, 6 weeks, rep1, cell46


title: iPS253-RPE, 6 weeks, rep1, cell45


title: THP-1_sgPOLD3-1_rep3


title: ER-Hoxa9_BRQ_6h_rep1

description:
RNAseq
BRQ_6h_rep1


title: ER-Hoxa9_BRQ_12h_rep1

description:
RNAseq
BRQ_12h_rep1


title: iPS253-RPE, 6 weeks, rep1, cell47


title: iPS253-RPE, 6 weeks, rep1, cell52


title: THP-1_sgPOLD3-1_rep2


title: ER-Hoxa9_BRQ_96h_rep1

description:
RNAseq
BRQ_96h_rep1


title: ER-Hoxa9_BRQ_6h_rep2

description:
RNAseq
BRQ_6h_rep2


title: ER-Hoxa9_BRQ_8h_rep2

description:
RNAseq
BRQ_8h_rep2


title: iPS253-RPE, 6 weeks, rep1, cell44


title: THP-1_sgPOLD3-1_rep1


title: ER-Hoxa9_BRQ_4h_rep2

description:
RNAseq
BRQ_4h_rep2


title: iPS253-RPE, 6 weeks, rep1, cell43


title: THP-1_sgMCM2-1_rep3


title: ER-Hoxa9_BRQ_4h_rep1

description:
RNAseq
BRQ_4h_rep1


title: iPS253-RPE, 6 weeks, rep1, cell42


title: THP-1_sgMCM2-1_rep4


title: ER-Hoxa9_BRQ_2h_rep2

description:
RNAseq
BRQ_2h_rep2


title: ER-Hoxa9_BRQ_2h_rep1

description:
RNAseq
BRQ_2h_rep1


title: ER-Hoxa9_BRQ_0h_rep1

description:
RNAseq
BRQ_0h_rep1


title: RagKO_8wk_3_ATGACAG

city: St. Louis

first: Aaron

last: Ver Heul

state: MO

address: 660 S Euclid Ave.

department: Medicine

institute: Washington University School of Medicine

description: gene expression

data_processing:
Basecalls and demultiplexing were performed with Illumina’s bcl2fastq2 software
RNA-sequencing reads were aligned to the Ensembl release 76 assembly with STAR v2.0.4b (Dobin et al., 2013). Gene counts were derived from the number of uniquely aligned unambiguous reads by Subread:featureCount v1.4.5 (Liao et al., 2014). Transcript counts were produced by Sailfish v0.6.3 (Patro et al., 2014). Sequencing performance was assessed for total number of aligned reads, total number of uniquely aligned reads, genes and transcripts detected, ribosomal fraction, known junction saturation, and read distribution over known gene models with RSeQC v2.3 (Wang et al., 2012).
Assembly: Mus_musculus reference build Ensembl_R76
Supplementary files format and content: Text files containing transcript counts quantified using Sailfish

source_name: skin-draining lymph node

growth_protocol: CD45.1 congenic mice were irradiated with 950cGy and then reconstituted with a 1:1 mixture of CD45.2 Rag1-/- and CD90.1 WT bone marrow from femurs and tibias 6 hours later. Chimeras received antibiotic drinking water from day -1 to day 7 post-transplant. Mice were kept for either 8 weeks or 20 weeks to allow full bone marrow reconstitution.

extract_protocol:
Type 2 innate lymphoid cells were sorted and stored in buffer RA1 plus TCEP -80C before processing following the manufacturer’s instructions using the NucleoSpin RNA XS kit (MACHEREY-NAGEL GmbH & Co).
Library preparation was performed with 1 ug of total RNA, and RNA integrity was determined using an Agilent Bioanalyzer. Ribosomal RNA was removed by a hybridization method using Ribo-ZERO kits (Illumina). mRNA was then fragmented in buffer containing 40 mM Tris acetate (pH 8.2), 100 mM potassium acetate, and 30 mM magnesium acetate and heated to 94C for 150 s. mRNA was reverse transcribed to yield cDNA using SuperScript III RT enzyme (Invitrogen, per manufacturer’s instructions) and random hexamers. A second strand reaction was performed to yield ds-cDNA. cDNA was blunt ended, had an A base added to the 30ends, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified for 12 cycles using primers incorporating unique index tags.

treatment_protocol: To expand the ILC2 population, mice were treated with rmIL-33 (R&D Systems, Minneapolis, MN). To increase expansion of cervical draining lymph nodes, 30 ng of IL-33 was injected into each cheek intradermally, while the remaining 240 ng was given intraperitoneally. These treatments were done daily for 7 days prior to harvest of lymph nodes for cell sorting.

tag: strain

value: Rag1 KO

tag: congenic marker

tag: genetic modification

value: WT/Rag KO bone marrow chimera

value: 8 weeks


title: THP-1_sgMCM2-1_rep2

tag: treatment duration


title: THP-1_sgMCM2-1_rep1


title: RagKO_8wk_2_TCCCAGC

description: gene expression


title: WT_8wk_3_TACAGAC

description: gene expression

value: WT


title: THP-1_sgGOT2-1_rep3


title: RagKO_20wk_3_TGTTTGT

description: gene expression

value: 20 weeks


title: iPS253-RPE, 6 weeks, rep1, cell41

data_processing:
Basecalls performed by Illumina Hiseq
Sequenced reads were trimmed for adaptor sequence, and were sorted by indexes using bcl2fastq
Reads were aligned to the gh38 genome assebly using STAR
Mapped reads seperated by genes, and the reads mapped for each gene were clusterd by molecular barcode
The number of cluteres for each gene was counted, which provides the number of molecules detected for each gene
Assembly: gh38
Supplementary files format and content: The calculated copy number of RNA moelcules per cell for each gene is listed.

value: 6 weeks


title: THP-1_sgGOT2-1_rep2


title: iPS253-RPE, 6 weeks, rep1, cell39


title: WT_8wk_1_CCGGACC

description: gene expression


title: RagKO_8wk_1_CTCCCGA

description: gene expression


title: iPS253-RPE, 6 weeks, rep1, cell38


title: ER-Hoxa9_BRQ_0h_rep2

description:
RNAseq
BRQ_0h_rep2


title: THP-1_sgNTC_rep3

value: sgNTC


title: THP-1_sgGOT2-1_rep1


title: WT_20wk_3_GGTCCTC

description: gene expression


title: RagKO_20wk_2_GCTTAGA

description: gene expression


title: iPS253-RPE, 6 weeks, rep1, cell37


title: iPS253-RPE, 6 weeks, rep1, cell36


title: WT_20wk_1_GGGTCAA

description: gene expression


title: WT_20wk_2_GTCGATA

description: gene expression


title: RagKO_20wk_1_AGATACT

description: gene expression


title: CUT&RUN_MCF7 FLAG-SMYD2 rep 3

city: Baltimore

first: Eneda

last: Toska

state: MD

address: 1650 Orleans Street, CRB1

institute: Johns Hopkins University

data_processing:
Bowtie2 -I 10 -X 700 -q --end-to-end --very-sensitive --no-unal --no-mixed --no-discordant --dovetail
bedtools v.2.31.0
Assembly: UCSC hg38
Supplementary files format and content: bigWig files containing normalized alignment results
Library strategy: CUT & RUN

growth_protocol: MCF7 cells were maintained in DMEM/F12 supplemented with 10% FBS and 1% penicillin streptomycin

extract_protocol:
Epicypher CUTANA CUT & RUN Kit
Epicypher CUTANA Library Prep Kit

treatment_protocol: Cells were transfected with empty vector or FLAG-SMYD2 using Lipofectamine 3000.  Media was changed after 24 hours and cells were collected 48 hours after transfection.

value: breast cancer


title: CUT&RUN_MCF7 Empty Vector rep 3

value: Empty Vector


title: CUT&RUN_MCF7 FLAG-SMYD2 rep 2


title: CUT&RUN_MCF7 FLAG-SMYD2 rep 1


title: THP-1_sgNTC_rep1


title: iPS253-RPE, 6 weeks, rep1, cell34

extract_protocol:
RNAs from cell lysate in each well was directly used for library preparation without purification
Libraries were prepared by using molecular barcoding technique. First, for control, the same amount of spike-in RNAs, a known number of External RNA Controls Consortium (ERCC) RNA molecules, was added into every tube which contains an isolated cell. Then, the cells were lysed and RNA molecules were fragmentated by temperature elevation. cDNAs were generated by reverse transcription using a reverse transcription primer, in which the first common primer sequence, molecular barcode, fourteen T bases, and one V (A or G or C) base are tandemly arranged from 5' end. In the reverse transcription, the second common primer sequence was also attached to 3' end of each generated cDNA molecule by template switching. Subsequently, cDNAs were amplified by PCR using two primers; one primer contains an illumina adapter sequence, sample index, and the first common primer sequence, and the other primer has another illumina adapter sequence, sample index, and the second common primer sequence.


title: Genomewide_THP1_BRQ_CD11b_rep2_down

data_processing:
FASTQ files were demultiplexed and barcode sequences were matched to the master list of CRISPRi barcodes. Only perfect matches were retained. Viability for each guide in the presence of brequinar was calculated by calculating a counts per million (CPM) value for each guide relative to the total library size in each sample, dividing the sum of CPMs from the CD11b up and down fractions by the CPM from the plasmid library, then taking its z-score relative to all other guides. Guide scores were calculated by taking the log2 ratio of CPMs between the CD11b-high and CD11b-low fraction for each guide, and the median score across all 10 guides for each gene was used as the gene score. Gene interactions were plotted for the top 500 scoring genes using STRING and Cytoscape, and GO annotations were calculated using Metascape.
Supplementary files format and content: Raw counts for each of the four cell fractions.

source_name: THP-1 cells

extract_protocol:
The CRISPRi screen was performed as described (Palmer et al., 2018) with the following modifications, using THP-1 dCas9 cells generated as above and a genome-wide library with 10 sgRNAs per gene (Addgene #1000000090). The library was electroporated into MegaX cells (Invitrogen #C640003) and plasmid was maxiprepped (Qiagen) and sequenced as described below to ensure even representation of guides. THP-1 cells were always maintained between 250,000 and 1 million cells/ml, and two biological replicates were performed starting at the lentiviral infection step. 200 million cells were infected for 24 hours with lentivirus at an MOI between 0.25 and 0.35 with 8 μg/ml polybrene. Lentiviral media was then replaced with fresh media, and 24 hours later, 1 μg/ml puromycin was added to select library-containing cells. Media was exchanged and fresh puromycin was added 24 and 48 hours afterwards. Four days after puromycin treatment, viability was >90% and BFP positivity was >97% for both replicates, indicating that the selection was complete. At this point, puromycin was removed and 200 million cells were treated with 500 nM brequinar. 48 hours later, cells were concentrated to 20 million cells/ml in fresh media, and at least 15 million cells from both the top and bottom 25% of CD11b expression were sorted using a BD FACSAria, pelleted, and frozen at -80ºC for later processing.
To obtain amplicons for sequencing, genomic DNA from each cell pellet was extracted using the Machery Nagel Nucleospin Blood L kit following the manufacturer’s instructions except with an overnight incubation at 70C and elution of DNA into 200 μl 10mM Tris (pH 8.5). Guides were then PCR amplified using a universal i7 primer and custom i5 primers indexed for each sample, with reactions split such that each 50 μl PCR reaction contained no more than 2.5μg DNA in a 20 μl volume. Most pellets were split across either 16 or 32 PCR reactions depending on cell number. PCR was performed using the NEBNext Ultra Q5 2X Master Mix with the following cycling parameters: initial denaturation at 98°C for 2 minutes, then 24 cycles of denaturation at 98°C for 15 seconds and annealing/extension at 65°C for 75 seconds, then a final extension of 65°C for 5 minutes. All reactions for each pellet were pooled, and successful amplification was verified by running 10 μl of the PCR on an agarose gel to obtain an expected band between 250 and 260bp. 200 μl of the PCR reaction (roughly corresponding to 1μg of amplicon) was then purified using a double-sided selection with AMpure XP beads (Beckman Coulter). First, to remove genomic DNA, 0.5X volume of beads (100 μl) was added to the PCR reaction, mixed well, and incubated at room temperature for 10 minutes, and beads were placed on a magnet and supernatant was saved. Second, to remove primer dimers, 135 μl beads were added to 290 μl of supernatant (final bead volume of 1.2X), mixed well, and incubated at room temperature for 10 minutes, and beads were placed on a magnet and supernatant was discarded. DNA was then eluted from the beads using 20 μl of 0.1X TE (pH 8.5), quantified using Qubit and Bioanalyzer, and pooled for 1x75 sequencing on an Illumina NextSeq 500 using an equimolar mix of custom read 1 primers (p5_seq_primer, listed in Table S5).

value: THP-1 dCas9-KRAB cells

value: Brequinar 500nM


title: iPS253-RPE, 6 weeks, rep1, cell40


title: WT_8wk_2_GCAACGC

description: gene expression


title: CUT&RUN_MCF7 Empty Vector rep 2


title: MCF7 KMT2D K1330A Clone 2 Alpelisib Rep3

data_processing:
salmon, v1.2.0
Assembly: hg18
Supplementary files format and content: .sf file including the transcript quantification results

growth_protocol: MCF7 cells were maintained in MEM supplemented with 1% Sodium Pyruvate, 1% GlutaMAX, 0.01 mg/mL human recombinant insulin, 10% FBS and 1% penicillin streptomycin

extract_protocol:
Qiagen RNeasy Mini Kit
Illumina

treatment_protocol: Cells were seeded and then treated 24 hours later with DMSO or 1µM alpelisib. Cells were collected 24 hours after drug treatment.

value: KMT2D K1330A Clone 2

value: Alpelisib


title: iPS253-RPE, 6 weeks, rep1, cell33

value: iPSC derived RPE cell

value: RPE


title: CUT&RUN_MCF7 Empty Vector rep 1


title: Genomewide_THP1_BRQ_CD11b_rep2_up


title: MCF7 KMT2D K1330A Clone 2 Alpelisib Rep2


title: Genomewide_THP1_BRQ_CD11b_rep1_down


title: MCF7 KMT2D K1330A Clone 2 Alpelisib Rep1


title: THP-1_sgNTC_rep2

data_processing:
Mouse and human data were mapped to mm10 and hg38 genomes, respectively, using STAR v2.7.8a and default parameters.
Reads that mapped to transcripts were counted using featureCounts v2.0.1 (with parameters -M -O --fraction --primary).
Supplementary files format and content: A tab-delimited file containing raw counts generated from featureCounts.


title: iPS253-RPE, 6 weeks, rep1, cell35


title: MCF7 KMT2D K1330A Clone 2 DMSO Rep3

value: DMSO


title: iPS253-RPE, 6 weeks, rep1, cell31


title: MCF7 KMT2D K1330A Clone 2 DMSO Rep2


title: MCF7 KMT2D K1330A Clone 2 DMSO Rep1


title: MCF7 KMT2D K1330A Clone 1 Alpelisib Rep3

value: KMT2D K1330A Clone 1


title: THP1_DMSO_96h_rep4

description:
RNAseq
thp1_thp1_dmso_005pct_96_4

value: DMSO 0.05percent


title: MCF7 KMT2D K1330A Clone 1 Alpelisib Rep2


title: iPS253-RPE, 6 weeks, rep1, cell30


title: iPS253-RPE, 6 weeks, rep1, cell32


title: THP1_DMSO_96h_rep3

description:
RNAseq
thp1_thp1_dmso_005pct_96_3


title: iPS253-RPE, 6 weeks, rep1, cell29


title: Genomewide_THP1_BRQ_CD11b_rep1_up


title: MCF7 KMT2D K1330A Clone 1 DMSO Rep3


title: THP1_DMSO_96h_rep2

description:
RNAseq
thp1_thp1_dmso_005pct_96_2


title: MCF7 KMT2D K1330A Clone 1 DMSO Rep2


title: iPS253-RPE, 6 weeks, rep1, cell27


title: THP1_DMSO_96h_rep1

description:
RNAseq
thp1_thp1_dmso_005pct_96_1


title: MCF7 WT KMT2D Alpelisib Rep3

value: WT KMT2D


title: MCF7 KMT2D K1330A Clone 1 DMSO Rep1


title: THP1_DMSO_48h_rep4

description:
RNAseq
thp1_thp1_dmso_005pct_48_4


title: MCF7 KMT2D K1330A Clone 1 Alpelisib Rep1


title: iPS253-RPE, 6 weeks, rep1, cell25


title: MCF7 WT KMT2D DMSO Rep3


title: MCF7 WT KMT2D Alpelisib Rep1


title: MCF7 WT KMT2D DMSO Rep2


title: iPS253-RPE, 6 weeks, rep1, cell24


title: MCF7 WT KMT2D DMSO Rep1


title: THP1_DMSO_48h_rep2

description:
RNAseq
thp1_thp1_dmso_005pct_48_2


title: THP1_DMSO_48h_rep1

description:
RNAseq
thp1_thp1_dmso_005pct_48_1


title: SET-2, WT, UM171, H3K27ac, Rep2 [Quantitative ChIP-seq]

last: Liau

address: 12 Oxford St.

department: Department of Chemistry and Chemical Biology

institute: Harvard University

data_processing:
library strategy: Quantitative ChIP-seq
Paired-end eads were aligned to a composite reference genome for dm6 and hg38 utilizing bowtie2 (version 2.3.2) according to the Spiker analysis framework (version 1.03).
The resulting .bam file is duplicate-marked and down-sampled to comparable coverage using picard (version 2.22.3).
The Spiker split_bam.py script was run to calculate scaling factors and to split reads into an alignment file containing reads specifically aligning to the hg38 genome (Spiker, version 1.0.3).
This alignment file was then converted into spike-in normalized bigwig file for further analysis and visualization utilizing the deepTools bamCoverage command.
Assembly: hg38
Supplementary files format and content: bigWig

extract_protocol:
Cells were fixed in 1% formaldehyde, washed, snap frozen, and stored at -80ºC. Sonication of crosslinked cells was calibrated such that DNA was sheared to between 300bp and 2000bp. Drosophila S2 chromatin (Active Motif) and spike-in antibody (Active Motif) were added following lysis and sonication but prior to immunoprecipitation.
ChIP DNA was used to generate sequencing libraries by end repair (End-It DNA repair kit, Epicentre), 3’ A base overhang addition via Klenow fragment (NEB), and ligation of barcoded sequencing adapters.

value: acute myeloid leukemia


title: iPS253-RPE, 6 weeks, rep1, cell28


title: SET-2, WT, UM171, H3K27ac, Rep1 [Quantitative ChIP-seq]

value: WT


title: iPS253-RPE, 6 weeks, rep1, cell23


title: iPS253-RPE, 6 weeks, rep1, cell22


title: SET-2, WT, DMSO, H3K27ac, Rep2 [Quantitative ChIP-seq]

value: Vehicle


title: THP1_BRQ_96h_rep4

description:
RNAseq
thp1_thp1_brq_500nM_96_4

extract_protocol:
1 million live cells were sorted using a BD FACSAria and placed on ice. Immediately following the sort, cells were centrifuged, pelleted, and flash frozen in liquid nitrogen and placed at -80C. Pellets were processed in groups of 24 using the Zymo RNA Miniprep Kit and quantified using the Invitrogen Quant-it RNA kit. RNA was then diluted to a concentration of 2 ng/ul and 5ul of each sample was transferred to a 96 well plate.
Poly-A enrichment and cDNA synthesis were performed in parallel as described in Soumilion et al. 2014, followed by library preparation using the Nextera XT kit and paired-end sequencing on an Illumina NextSeq 500.


title: iPS253-RPE, 6 weeks, rep1, cell26

value: 6 weeks


title: SET-2, WT, DMSO, H3K27ac, Rep1 [Quantitative ChIP-seq]


title: SET-2, NΔ2, GSKLSD1, H3K27ac, Rep2

data_processing:
Paired-end reads were aligned to the GRCh38 human reference using the STAR (version 2.7.0e) aligner with splicing disabled.
The resulting .bam file is duplicate-marked and down-sampled to comparable coverage using picard (version 2.22.3).
Peaks for each replicate were called separately with HOMER (version 4.10) with matched inputs before establishing the final peak list with an irreproducible discovery rate (IDR) of 2%.
Replicates were then merged and converted into bigwig files for visualization and quantification with deepTools2
Assembly: hg38
Supplementary files format and content: bigWig and/or bed (except for Input sample)

extract_protocol:
SET-2 cells were fixed in 1% formaldehyde, washed, snap frozen, and stored at -80ºC. Sonication of crosslinked cells was calibrated such that DNA was sheared to between 300bp and 2000bp.
ChIP DNA was used to generate sequencing libraries by end repair (End-It DNA repair kit, Epicentre), 3’ A base overhang addition via Klenow fragment (NEB), and ligation of barcoded sequencing adapters.

treatment_protocol: Cells were seeded at 300,000 cells/ml and then treated with vehicle (0.1% DMSO) or 100 nM GSK-LSD1 for the specified time.

value: Anti-H3K27ac, Active Motif, 39133


title: SET-2, NΔ2, DMSO, H3K27ac, Rep1


title: iPS253-RPE, 6 weeks, rep1, cell21


title: SET-2, NΔ2, GSKLSD1, LSD1, Rep1

value: Anti-BHC110/LSD1, Bethyl, A300-215


title: THP1_BRQ_96h_rep3

description:
RNAseq
thp1_thp1_brq_500nM_96_3


title: MCF7 WT KMT2D Alpelisib Rep2

value: breast cancer


title: THP1_BRQ_96h_rep1

description:
RNAseq
thp1_thp1_brq_500nM_96_1


title: iPS253-RPE, 6 weeks, rep1, cell20


title: SET-2, NΔ2, DMSO, H3K27ac, Rep2


title: iPS253-RPE, 6 weeks, rep1, cell18


title: SET-2, NΔ2, GSKLSD1, LSD1, Rep2


title: THP1_BRQ_96h_rep2

description:
RNAseq
thp1_thp1_brq_500nM_96_2


title: THP1_BRQ_48h_rep4

description:
RNAseq
thp1_thp1_brq_500nM_48_4


title: SET-2, WT, GSKLSD1, RUNX1, Rep2

value: Anti-RUNX1, Santa Cruz, sc-36544


title: SET-2, NΔ2, DMSO, Input


title: iPS253-RPE, 6 weeks, rep1, cell19


title: SET-2, NΔ2, GSKLSD1, H3K27ac, Rep1


title: SET-2, NΔ2, DMSO, LSD1, Rep2


title: SET-2, WT, GSKLSD1, RUNX1, Rep1


title: THP1_DMSO_48h_rep3

description:
RNAseq
thp1_thp1_dmso_005pct_48_3


title: SET-2, WT, DMSO, RUNX1, Rep2


title: SET-2, WT, GSKLSD1, CEBPA, Rep2

value: Anti-CEBPA, Cell Signaling Technology, #8178


title: SET-2, NΔ2, GSK-LSD1, Input


title: iPS253-RPE, 6 weeks, rep1, cell14


title: SET-2, NΔ2, DMSO, LSD1, Rep1


title: THP1_BRQ_48h_rep3

description:
RNAseq
thp1_thp1_brq_500nM_48_3

value: Brequinar 500nM


title: SET-2, WT, GSKLSD1, CEBPA, Rep1


title: iPS253-RPE, 6 weeks, rep1, cell17


title: iPS253-RPE, 6 weeks, rep1, cell16


title: THP1_HU_96h_rep4

description:
RNAseq
thp1_thp1_hu_100uM_96_4

value: Hydroxyurea 100uM


title: SET-2, WT, DMSO, CEBPA, Rep2


title: iPS253-RPE, 6 weeks, rep1, cell15

value: iPSC derived RPE cell


title: SET-2, WT, DMSO, RUNX1, Rep1


title: iPS253-RPE, 6 weeks, rep1, cell13

value: RPE


title: THP1_BRQ_48h_rep2

description:
RNAseq
thp1_thp1_brq_500nM_48_2


title: THP1_BRQ_48h_rep1

description:
RNAseq
thp1_thp1_brq_500nM_48_1


title: iPS253-RPE, 6 weeks, rep1, cell12


title: THP1_HU_96h_rep2

description:
RNAseq
thp1_thp1_hu_100uM_96_2


title: SET-2, WT, DMSO, PU.1, Rep2

value: Anti-PU.1, Cell Signaling Technology, #2258


title: THP1_HU_96h_rep1

description:
RNAseq
thp1_thp1_hu_100uM_96_1


title: iPS253-RPE, 6 weeks, rep1, cell11


title: SET-2, WT, GSKLSD1, PU.1, Rep1


title: iPS253-RPE, 6 weeks, rep1, cell10


title: SET-2, WT, DMSO, RCOR1, Rep2


title: SET-2, WT, DMSO, PU.1, Rep1


title: SET-2, WT, GSKLSD1, H3K27ac, Rep2


title: SET-2, WT, DMSO, RCOR1, Rep1


title: THP1_HU_48h_rep4

description:
RNAseq
thp1_thp1_hu_100uM_48_4


title: iPS253-RPE, 6 weeks, rep1, cell9


title: THP1_HU_96h_rep3

description:
RNAseq
thp1_thp1_hu_100uM_96_3


title: SET-2, WT, GSKLSD1, PU.1, Rep2


title: SET-2, WT, GSKLSD1, H3K27ac, Rep1


title: SET-2, WT, DMSO, H3K27ac, Rep2


title: THP1_HU_48h_rep3

description:
RNAseq
thp1_thp1_hu_100uM_48_3


title: SET-2, WT, DMSO, CEBPA, Rep1


title: iPS253-RPE, 6 weeks, rep1, cell8


title: iPS253-RPE, 6 weeks, rep1, cell7


title: THP1_HU_48h_rep2

description:
RNAseq
thp1_thp1_hu_100uM_48_2


title: Shoot, 30D, anti-Myc

city: Kunming

first: Lirong

last: Zhao

state: Yunnan

address: No.88 Xuefu Road

department: Xishuangbanna Tropical Botanical Garden

institute: Chinese Academy of Sciences

data_processing:
The ChIP library underwent quality assessment based on the following criteria: NRF>0.9, PBC1>0.9, and PBC2>3.
The raw sequencing reads were subjected to quality filtering using Trimmomatic (version 0.36) and aligned to the rice reference genome (rice.uga.edu, version 7.0) using bwa (0.7.12-r1039) with default parameters. The peak analysis was conducted using MACS2 (2.2.7.1) with the parameters "-B -f BAMPE -q 0.05".
Assembly: Oryza sativa genome (rice.uga.edu, version 7.0)
Supplementary files format and content: peak

organism: Oryza sativa

source_name: Shoot

extract_protocol:
A total of 2 g of seedling shoots from transgenic pUbi:OsWRKY11-Myc plants were harvested and fixed in a crosslinking solution containing 1% (w/v) formaldehyde under vacuum for 30 min on ice. crosslinking was terminated by treating with 0.125 mM glycine under vacuum for 5 min.
ChIP assay was performed using the EpiQuik™ Plant ChIP Kit (Epigentek).

value: Shoot

value: transgenic pUbi:OsWRKY11-Myc

value: anti-Myc (Abmart)


title: Shoot, 30D Input


title: SET-2, WT, GSKLSD1, GFI1B, Rep2

value: acute myeloid leukemia

value: Anti-GFI-1B, Santa Cruz, sc-28356